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In low risk MDS, increased apoptosis of erythroid progenitors mediated via CD95 (Fas) activation has been described to result in peripheral cytopenia. Blockade of the CD95 system can improve erythropoiesis in MDS. Asunercept (APG101) is a fusion protein consisting of the extracellular domain of human CD95 and the Fc domain of human IgG1 blocking the interaction between CD95 and its ligand. Here we report on results from a phase I study in 20 transfusion-dependent low and intermediate risk MDS patients treated with intravenous asunercept (EudraCT 2012-003027-37). Primary objectives were safety and tolerability as well as pharmacodynamic effects. Secondary objectives were hematologic improvement, incidence and time to leukemic progression as well as overall survival. Frequency and severity of adverse events were in range of what could be expected in a patient cohort comprising of elderly MDS patients. Two patients experienced a serious adverse event with a suspected relationship to asunercept. The incidence of disease progression was low. In the 20 patients a decrease of the transfusion need from a mean of 10,8 (±5,1) pRBCs during the 12 weeks treatment phase to a mean of 10,0 (±4,2) pRBCs at the end of the study was observed. In conclusion, asunercept was well tolerated and showed efficacy in transfusion-dependent low and intermediate risk MDS patients. Further clinical investigation is warranted, particularly in combination with erythropoiesis stimulating agents (ESAs). [ABSTRACT FROM AUTHOR]

Summary Background & aims Azacitidine (AZA) therapy has become the recommended first-line treatment for patients with high-risk myelodysplastic syndromes (MDS) and oligoblastic (<30% bone marrow blasts) acute myeloid leukemia (AML). However, improvement of the efficacy of AZA treatment remains a challenge. We retrospectively tested the hypothesis that VitD levels (25-hydroxyvitamin D3) prior to start of first-line AZA therapy are predictive of overall survival (OS) in patients diagnosed with MDS and secondary oligoblastic AML. Furthermore, the antiproliferative effects of AZA in combination with 25-hydroxyvitamin D3 and 1α,25-dihydroxyvitamin D3 were investigated in vitro . Methods A total of 58 patients treated at our center between 2006 and 2014 were analyzed. Serum levels of VitD were quantified using a standard, commercially available 25-hydroxyvitamin D3 chemiluminescent immunoassay. Effects on cell proliferation were assessed using tetrazolium-based MTT assays. Results Median serum VitD level prior to AZA treatment was 32.8 nM (range 11.0–101.5 nM). Patient, disease and treatment characteristics did not differ significantly between the low (≤32.8 nM; n = 29) and high (>32.8 nM; n = 29) VitD group. Estimated probability of 2-year OS in the low versus high VitD group was 14% versus 40% (P < 0.05). In multivariable analysis with OS as endpoint, adverse cytogenetics (HR 2.66, P = 0.03) and VitD (per 10 nM decrease, HR 1.68, P = 0.02) were independent predictors of worse survival. In-vitro treatment of myeloid cell lines with AZA in combination with VitD produced synergistic and additive antiproliferative effects. Addition of nanomolar VitD concentrations to AZA resulted in potentiation of AZA activity. Conversely, combination with the VitD antagonist TEI-9647 resulted in inhibition of AZA activity. Conclusions Our study suggests that higher VitD levels were associated with a survival advantage following first-line AZA therapy. Enhanced cytotoxic effects upon combination treatment may contribute to the observed clinical effects. VitD repletion/supplementation during AZA treatment should be explored. [ABSTRACT FROM AUTHOR]

Neutralizing anti-drug antibodies (NAbs) against therapeutic interferon beta (IFNβ) in people with multiple sclerosis (MS) are measured with cell-based bioassays. The aim of this study was to redevelop and validate two luciferase reporter-gene bioassays, LUC and iLite, using a cut-point approach to identify NAb positive samples. Such an approach is favored by the pharmaceutical industry and governmental regulatory agencies as it has a clear statistical basis and overcomes the limitations of the current assays based on the Kawade principle. The work was conducted following the latest assay guidelines. The assays were re-developed and validated as part of the “Anti-Biopharmaceutical Immunization: Prediction and analysis of clinical relevance to minimize the risk” (ABIRISK) consortium and involved a joint collaboration between four academic laboratories and two pharmaceutical companies. The LUC assay was validated at Innsbruck Medical University (LUC IMU ) and at Rigshospitalet (LUC RH ) Copenhagen, and the iLite assay at Karolinska Institutet, Stockholm. For both assays, the optimal serum sample concentration in relation to sensitivity and recovery was 2.5% ( v / v ) in assay media. A Shapiro–Wilk test indicated a normal distribution for the majority of runs, allowing a parametric approach for cut-point calculation to be used, where NAb positive samples could be identified with 95% confidence. An analysis of means and variances indicated that a floating cut-point should be used for all assays. The assays demonstrated acceptable sensitivity for being cell-based assays, with a confirmed limit of detection in neat serum of 1519 ng/mL for LUC IMU , 814 ng/mL for LUC RH , and 320 ng/mL for iLite. Use of the validated cut-point assay, in comparison with the previously used Kawade method, identified 14% more NAb positive samples. In conclusion, implementation of the cut-point design resulted in increased sensitivity to detect NAbs. However, the clinical significance of these low positive titers needs to be further evaluated. [ABSTRACT FROM AUTHOR]

• ECP was able to increase Tfh cells to ameliorate GvHD. • Upregulation of Th22 cells was observed in aGvHD patients with CR. • Tim-3 expression was downregulated on effector T cells by ECP. • ECP shows immunomodulatory effects in GvHD setting. Extracorporeal photopheresis (ECP), a personalized cellular immunotherapy, constitutes a promising treatment for steroid-refractory/-resistant graft-versus-host disease (SR-GvHD), with encouraging clinical response rates. To further investigate its mechanism of action, ECP's effects on T helper (Th) cells as well as on expression of immune checkpoint (PD-1 and Tim-3) and apoptotic (Fas receptor [FasR]) molecules were investigated in 27 patients with SR-GvHD. Our data show that GvHD patients had significantly higher levels of Th2, Th17, Th22 and granulocyte-macrophage colony-stimulating factor (GM-CSF)-positive Th (ThG) cells and clearly lower levels of T follicular helper (Tfh) cells, including Th1- and Th2-like cells, compared with healthy donors. ECP therapy for GvHD was effective through the modulation of different Th subsets: increases of Th22 (1.52-fold) and Tfh cells (1.48-fold) in acute GvHD (aGvHD) and increases of Th2-like Tfh cells (1.74-fold) in chronic GvHD (cGvHD) patients were associated with clinical response. Expression of FasR was further upregulated in CD4+CD8+ T cells. Additionally, Tim-3–expressing effector T cells associated with the severity of GvHD were reduced. Taken together, these data show that ECP therapy exerts immunomodulatory effects by promoting a balanced immune reconstitution and inducing immune tolerance. Therefore it represents an attractive option for the treatment of GvHD. [ABSTRACT FROM AUTHOR]

Abstract: Sequencing of the acute myeloid leukemia genome revealed somatic mutations in isocitrate dehydrogenase-1. Acute myeloid leukemia frequently develops from myelodysplastic syndrome. In order to test whether myelodysplastic syndrome also carries isocitrate dehydrogenase-1 mutations, we stained a series of bone marrow samples from patients with myelodysplastic syndrome using an antibody specific for the R132H mutation. Three out of 71 patients exhibited antibody binding to myeloid precursor cells. The presence of the R132H mutation was confirmed by DNA sequencing. We demonstrated that isocitrate dehydrogenase-1 mutations occur in myelodysplasia preceding acute myeloid leukemia and that the R132H alteration can be detected by immunohistochemistry. [Copyright &y& Elsevier]

Inhibition of Janus-activated kinase-1 (JAK1) is a promising clinical concept for post-transplant immunosuppression and autoimmunity. However, it also raises concerns regarding possible immunosuppressive side effects. Our study investigates JAK1 signalling in the context of CD40L and bacterially activated human MoDC using siRNA and biological inhibitors. We demonstrate that strong stimuli (e.g. intact Escherichia coli or LPS in addition to IL-1β) induce IL-12p70 via a ROS/RELA/CDK9 pathway that is inhibited by simultaneous JAK1/STAT3 signalling. Transcription is effective if RELA recruits the positive transcription elongation factor b (P-TEFb) component CDK9 to a combined RELA/STAT3 binding site −50 to −20 bp upstream of the start site of the IL-12p35 promoter. STAT3 simultaneously attaches to this site and inhibits CDK9 binding. In the presence of IFNγ, JAK1/2 inhibitors block STAT1/IRF1/IRF8-dependent activation and simultaneously enhance CDK9-dependent activation signals. This inverse regulation of IFNγ- vs. E. coli -induced cytokine production by JAK inhibitors including Ruxolitinib was similarly observed for IL-6 and TNF-α production, but not for IL-10 production. Thus, JAK1 inhibition enhances IL-12p70 production in this context by increased DNA binding of CDK9. In contrast, weak RELA-activation signals (CD40L, LPS) depended on IFN-γ induced STAT1/IRF1/IRF8 co-signalling, which was completely blocked by JAK inhibitors as reported before. Our results suggest a novel molecular mechanism of how cytokine responses to invading pathogens are separable from IFNγ-dependent autoimmunity by targeting JAK1/STAT3 activation. [ABSTRACT FROM AUTHOR]

To expand the current knowledge about azacitidine (Aza) and donor lymphocyte infusions (DLI) as salvage therapy for relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and to identify predictors for response and survival, we retrospectively analyzed data of 154 patients with acute myeloid leukemia (AML, n = 124), myelodysplastic (MDS, n = 28), or myeloproliferative syndrome (n = 2). All patients received a median number of 4 courses of Aza (range, 4 to 14) and DLI were administered to 105 patients (68%; median number of DLI, 2; range, 1 to 7). Complete and partial remission rates were 27% and 6%, respectively, resulting in an overall response rate of 33%. Multivariate analysis identified molecular-only relapse (hazard ratio [HR], 9.4; 95% confidence interval [CI], 2.0 to 43.5; P = .004) and diagnosis of MDS (HR, 4.1; 95% CI, 1.4 to 12.2; P = .011) as predictors for complete remission. Overall survival (OS) at 2 years was 29% ± 4%. Molecular-only relapse (HR, .14; 95% CI, .03 to .59; P = .007), diagnosis of MDS (HR, .33; 95% CI, .16 to .67; P = .002), and bone marrow blasts <13% (HR, .54; 95% CI, .32 to .91; P = .021) were associated with better OS. Accordingly, 2-year OS rate was higher in MDS patients (66% ± 10%, P = .001) and correlated with disease burden in patients with AML. In summary, Aza and DLI is an effective and well-tolerated treatment option for patients with relapse after allo-HSCT, in particular those with MDS or AML and low disease burden. The latter finding emphasizes the importance of stringent disease monitoring and early intervention. [ABSTRACT FROM AUTHOR]

Abstract: There is accumulating evidence indicating that endothelial factors are involved in the pathogenesis of GVHD. We have recently shown that steroid-refractory, but not sensitive, GVHD is characterized by higher pretransplantation serum levels of angiopoetin-2 (ANG2), a hormone mediating endothelial vulnerability. To evaluate whether endothelial vulnerability is a risk factor for GVHD per se or becomes important only when noticeable GVHD is established, we measured ANG2 along with additional serum markers of endothelial stress, including soluble thrombomodulin (sTM), IL-8 (CXCL8), and hepatocyte growth factor (HGF), in patients with no, low-grade, or severe GVHD. Patients with refractory GVHD exhibited elevated serum levels of ANG2, sTM, HGF, and IL-8 posttransplantation compared with patients with sensitive GVHD and patients without GVHD. Pretransplantation ANG2 was the only growth factor correlated with the risk of refractoriness and mortality, and then only within the subset of patients who developed grade III-IV GVHD. In contrast, ANG2 was not predictive of GVHD or nonrelapse mortality (NRM) in patients with no GVHD or low-grade GVHD. These findings provide evidence that endothelial function plays an important role in the pathogenesis of steroid refractoriness in ongoing GVHD; however, endothelial vulnerability does not predict incidence of GVHD. [Copyright &y& Elsevier]

Abstract: CD40Ligand (CD40L) represents a strong endogenous danger signal associated with chronic inflammatory disease. CD40L induces activation of antigen-presenting cells (APCs) such as DCs, monocytes, B-cells and endothelial cells. However, CD40 activation alone, whilst inducing IL-10 production, is insufficient to induce interleukin (IL)-12p70 release in human APCs suggesting that additional cytokine signals (e.g. GM-CSF, IL-4 or IFN-γ) are required for the induction of a pro-inflammatory cytokine profile. We demonstrate that IFN-γ-induced Janus kinase 1 (JAK1) enhances CD40-induced IL-12p70 release whilst simultaneously inhibiting IL-10 synthesis, resulting in a pro-inflammatory phenotype of CD40L-activated dendritic cells (DCs). JAK2 mediated enhancing effects on IL-12p70 but did not inhibit IL-10 release, whereas Tyk2 mediated inhibitory effects on IL-12p70 release in this system. The mechanism by which complementary IFN-γ/JAK activities affect IL-12p70 production involves STAT1 activation and de novo induction of interferon-responsive factors (IRF)-1 and IRF-8. Simultaneously, JAK1 was unique in inhibiting IL-10 synthesis via STAT1 and IRF-8 with both transcription factors binding to the IL-10 promoter. We demonstrate that CD40- and JAK/STAT/IRF-signalling pathways are strictly complementary for the induction of a pro-inflammatory cytokine profile in human APCs. This suggests that a number of CD40 effects in chronic inflammatory diseases might be weakened by targeting JAK/STAT. [ABSTRACT FROM AUTHOR]

Objective: Active vaccination in the allogeneically reconstituted tumor-bearing host essentially requires donor T–cell tolerance. To create a basis for vaccination in the allogeneically reconstituted, lymphoma-bearing host, we elaborate a reconstitution protocol that supports thymus repopulation and tolerance induction. Methods: Myeloreductively conditioned, lymphoma-bearing mice were vaccinated after reconstitution with hematopoietic progenitor cells. Readout systems included recovery of donor-derived T cells, graft vs host disease (GVHD), anti-host and anti-lymphoma cytotoxicity, as well as tumor growth rate and tumor rejection. Results: In tumor-free mice, myeloreductive conditioning, together with natural killer cell depletion of the host and transfer of T cell–depleted bone marrow cells, allows reconstitution without severe GVHD. However, in hematological malignancies, donor-derived T-progenitor cells hardly immigrated into the thymus. As a consequence, the frequency of severe GVHD was significantly increased, which prohibited active vaccination. Thymus repopulation became improved by strengthening myeloreductive conditioning; by supporting thymocyte expansion via interleukin-7; and, most strongly, by a small dose of donor-derived CD4+CD8+ thymocytes, which preferentially homed into the thymus. Active vaccination, in combination with this reconstitution protocol, did not strengthen GVHD, but significantly improved survival time and survival rate of lymphoma-bearing mice. Conclusion: The negative impact of hematological malignancies on thymus repopulation and central tolerance induction can, at least in part, be corrected by application of a small number of donor-derived T-progenitor cells. [Copyright &y& Elsevier]

Abstract: Background: Recent retrospective studies demonstrate that elderly persons have a shortened protection period following vaccination with recall antigens. Methods: We now analysed the effect of booster vaccination with a multivalent vaccine containing tetanus, dipththeria, pertussis and polio antigens in 252 healthy elderly persons. The magnitude of the humoral immune response was assessed by antibody measurements. Results: Comparison with a small control group of 21 younger persons demonstrates that pre- and post-vaccination antibody concentrations are lower in elderly persons for all antigens except polio, for which higher pre- and similar post-vaccination antibody levels are observed. Using multiple linear regression analysis we also show that the magnitude of the humoral immune response in elderly persons greatly depends on pre-vaccination antibody concentrations in the case of tetanus, diphtheria and pertussis, but much less so in the case of polio, against which priming and preceding booster immunizations were performed with attenuated live vaccine. Conclusion: Regular booster vaccinations throughout life are of clinical importance to maintain the ability to respond to recall antigens in old age. Longer lasting protection and good responsiveness to boosting in spite of low antibody titres can be expected following exposure to live vaccine earlier in life. [Copyright &y& Elsevier]

Immunostimulating complex (ISCOM®) vaccines are particulate antigen delivery vehicles composed of saponin, cholesterol, phospholipid and immunogen. Here we illustrate that ISCOM®-based vaccines represent an attractive modality for the development of anti-cancer vaccines. Using murine models and a model cancer antigen, ISCOM® vaccines were shown to induce potent CD8 T cell responses, to mediate protection in three different tumor models, to promote Th1-biased immunity, and to induce CD8 T cell responses in the absence of CD4+ T cell help. The former three activities were also found to be substantially improved when the vaccine antigen was associated with the ISCOM® structure. Furthermore, the presence in vivo of pre-existing antibodies against the vaccine antigen did not inhibit CD8 T cell induction by the ISCOM® vaccine. Although vaccination was effective against challenge with vaccine-antigen expressing tumors, no activity against neighboring vaccine-antigen negative tumor cells was observed, indicating that determinant spreading or bystander activity does not lead to significant anti-cancer activity. [Copyright &y& Elsevier]

Allogeneic stem cell transplantation (SCT) is potentially curative therapy for patients with MDS. Prior studies have shown no overall-survival (OS) advantage of any conditioning regimen over the others. Reduced-intensity conditioning (RIC) is usually associated with lower non-relapse mortality (NRM) compared to myeloablative conditioning (MAC), however, relapse rates are higher, resulting in similar OS. Fludarabine and treosulfan (FT) is a reduced-toxicity regimen with intense anti leukemia activity in patients with myeloid malignancies. We explored outcomes following FT conditioning in comparison with other regimens. We retrospectively analyzed SCT outcomes of MDS patients reported to the chronic malignancies working party of EBMT (n=1722). The conditioning regimens were defined according to standard EBMT criteria and included FT (n=367), RIC (n=687) or MAC (n=668). FT and RIC recipients were older than MAC recipients, median age 59, 59 and 51 years, respectively (P < 0.001). More FT recipients had untreated MDS at SCT, 45%, 26% and 36%, respectively (P < 0.001). Disease status at diagnosis, cytogenetics, secondary origin and prior transformation to AML, were not different between the regimens. With a median follow-up of 20 months (1-171), 807 patients are alive, 328 died of relapse and 587 had NRM. The 5-year rates of relapse, NRM and OS for the entire group were 21% (95%CI, 19-23), 35% (95%CI, 33-38) and 44% (95%CI, 42-437), respectively. Relapse rates were similar after FT and MAC, 16% (13-21) and 19% (16-22), respectively, significantly lower than after RIC, 25% (21-28) (P = 0.003). Multivariate analysis (MVA) identified age >55 years (HR 1.42), advanced histology (RAEB 1/2, HR 2.01/ 1.73), prior transformation to AML (HR 1.87), refractory disease to prior therapies (HR 1.75) secondary origin (HR 1.33) and poor cytogenetics (HR 2.83) as associated with higher relapse risk. FT conditioning was associated with a lower risk (HR 0.63, P = 0.006). NRM was lower after FT and RIC than after MAC, 34% (29-39), 33% (30-37) and 38% (35-40), respectively (P = 0.05). MVA identified age >55 years (HR 1.39) and MAC (HR 1.34) as associated with higher NRM, while SCT from sibling donor (HR 0.81) and in CR (HR 0.80) were associated with a lower risk. In all, 5-year OS was 50% (44-55), 43% (38-47), and 43% (39-47), after FT, RIC and MAC, respectively (Figure 1, P = 0.03). MVA identified age >55 years (HR 1.39), advanced histology (RAEB 1/2, HR 1.35/ 1.10), prior transformation to AML (HR 1.37), and poor cytogenetics (HR 1.58) as associated with lower OS. FT conditioning was associated with better OS (HR 0.79, P = 0.01). FT is associated with similar low relapse rates as MAC and similar low NRM as RIC, resulting in improved outcome over both RIC and MAC. FT might be the preferred regimen for SCT in MDS. These observations merit further study in randomized prospective trials. [ABSTRACT FROM AUTHOR]