ALBERT

Description: Transcription factors of the NFAT (nuclear factor of activated T cells) family regulate the expression of many genes encoding immunoregulatory cytokines and cell surface proteins during the immune response. The NFAT protein NFAT1 (NFATp) is expressed and functional in T cells, B cells, mast cells, and natural killer cells. Here we report a detailed analysis of the enhanced eosinophil responses of NFAT1-deficient mice, observed in an in vivo model of allergic inflammation. In addition to the pleural eosinophilia described previously, NFAT1−/− mice that have been sensitized with antigen display a significant increase, relative to wild-type mice, in the numbers of eosinophils in bone marrow and peripheral blood. After restimulation with antigen in vitro, antigen-responsive T cells from the draining lymph nodes of NFAT1−/− mice show increased expression of mRNA encoding the Th2 cytokines interleukin-4 (IL-4), IL-5, and IL-13. Consistent with this finding, there is a pronounced increase in the levels of IL-5 and IL-13 in the pleural cavities of sensitized NFAT1−/− mice after allergen challenge in vivo. Furthermore, development of eosinophilia depends on overexpression of IL-4 and IL-5, because it is strongly inhibited by administration of neutralizing antibodies to either of these cytokines. These results indicate that NFAT1-deficient mice are prone to develop a classically allergic phenotype characterized by eosinophilia and increased production of Th2 cytokines. Thus, the presence of NFAT1 might inhibit the allergic response, perhaps by interfering with the development of Th2 immune responses, and the lack or dysfunction of NFAT1 could potentially underlie certain cases of atopic disease.

Description: Transcription factors of the nuclear factor of activated T cells (NFAT) family are thought to regulate the expression of a variety of inducible genes such as interleukin-2 (IL-2), IL-4, and tumor necrosis factor-α. However, it remains unresolved whether NFAT proteins play a role in regulating transcription of the interferon- γ (IFN-γ) gene. Here it is shown that the transcription factor NFAT1 (NFATc2) is a major regulator of IFN-γ production in vivo. Compared with T cells expressing NFAT1, T cells lacking NFAT1 display a substantial IL-4–independent defect in expression of IFN-γ mRNA and protein. Reduced IFN-γ production by NFAT1−/−× IL-4−/− T cells is observed after primary in vitro stimulation of naive CD4+ T cells, is conserved through at least 2 rounds of T-helper cell differentiation, and occurs by a cell-intrinsic mechanism that does not depend on overexpression of the Th2-specific factors GATA-3 and c-Maf. Concomitantly, NFAT1−/−× IL-4−/− mice show increased susceptibility to infection with the intracellular parasiteLeishmania major. Moreover, IFN-γ production in a murine T-cell clone is sensitive to the selective peptide inhibitor of NFAT, VIVIT. These results suggest that IFN-γ production by T cells is regulated by NFAT1, most likely at the level of gene transcription.

Description: Transcription factors of the NFAT (nuclear factor of activated T cells) family regulate the expression of many genes encoding immunoregulatory cytokines and cell surface proteins during the immune response. The NFAT protein NFAT1 (NFATp) is expressed and functional in T cells, B cells, mast cells, and natural killer cells. Here we report a detailed analysis of the enhanced eosinophil responses of NFAT1-deficient mice, observed in an in vivo model of allergic inflammation. In addition to the pleural eosinophilia described previously, NFAT1−/− mice that have been sensitized with antigen display a significant increase, relative to wild-type mice, in the numbers of eosinophils in bone marrow and peripheral blood. After restimulation with antigen in vitro, antigen-responsive T cells from the draining lymph nodes of NFAT1−/− mice show increased expression of mRNA encoding the Th2 cytokines interleukin-4 (IL-4), IL-5, and IL-13. Consistent with this finding, there is a pronounced increase in the levels of IL-5 and IL-13 in the pleural cavities of sensitized NFAT1−/− mice after allergen challenge in vivo. Furthermore, development of eosinophilia depends on overexpression of IL-4 and IL-5, because it is strongly inhibited by administration of neutralizing antibodies to either of these cytokines. These results indicate that NFAT1-deficient mice are prone to develop a classically allergic phenotype characterized by eosinophilia and increased production of Th2 cytokines. Thus, the presence of NFAT1 might inhibit the allergic response, perhaps by interfering with the development of Th2 immune responses, and the lack or dysfunction of NFAT1 could potentially underlie certain cases of atopic disease.

Description: Beyond disease biology, the success of allogeneic hematopoietic stem cell transplantation (HSCT) in patients with hematological malignancies is mainly determined by the occurrence and extent of graft versus host disease (GVHD). Therefore, prevention of GVHD is the major goal and challenge in clinical HSCT. A calcineurin-inhibitor combined with methotrexate is the standard graft versus host disease (GVHD) prophylaxis after allogeneic hematopoietic stem cell transplantation (HSCT). Everolimus is a derivative of sirolimus, which also seems to mediate anti-leukemia effects. Given the potential synergism and favourable toxicity profile of everolimus and tacrolimus (EVTAC) after allogeneic HSCT we sought to investigate the efficacy of this combination in patients with either myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). We report a combination of everolimus (days 0–56) and Tacrolimus (from day −1 on) in 24 patients (pts, median age 62 years) with either MDS (n=17) or AML (n=7) undergoing intensive busulfan-based conditioning followed by HSCT from related (n=4) or unrelated matched (n=12) or 1-allele mismatched (n=8) donors. All pts engrafted and only one experienced grade IV mucositis. However, although everolimus was scheduled to be administered up to day 56, patients received the drug a median of 44 days (range 10–56) only. The reason for premature discontinuation (50%) were either occurrence of early-onset (day 6) GVHD associated hyperbilirubinemia CTC grade 4 (n=1), transplantation-associated microangiopathy (TMA, n=3), sinusoidal obstructive syndrome (SOS) of the liver (n=6) or a drop of platelets after engraftment by at least 50% (n=2). Nine pts (37%) developed grade II–IV acute GVHD, however, chronic extensive GVHD was observed in 11 of 17 (64%) evaluable pts. TMA occurred in 7 pts (29%) with two cases of acute renal failure. In five out of seven patients with TMA either tacrolimus (n=4) or everolimus (n=1) blood through levels were slightly above the upper target level at the time of TMA appearance. The study was terminated prematurely because additional 6 pts (25%) developed SOS, which was fatal in two cases. With a median follow-up of 26 months, the 2-year overall survival rate is 47%. In conclusion, although this new combination appears to be effective as prophylactic regimen for acute GVHD, the incidence of TMA and SOS seems to be higher compared to other regimens. As a result this combination cannot be recommended as prophylactic regimen after busulfan-based intensive conditioning. However, studies in the context of TBI-based or reduced-intensity conditioning regimens might come to a different conclusion.

Description: Abstract 202 Introduction: In a completed study (NCT00337519), patients with advanced B-CLL received allogeneic stem cell transplantation (SCT) after cytoreductive treatment with alemtuzumab followed by a wash-out period for the antibody and conditioning with fludarabine/busulfan. Aim of the present investigation was to correlate flow cytometric levels of minimal residual disease (MRD) in the peripheral blood at different time points after transplantation with patient outcome. Patients and Methods: In 58 CLL patients 900 flow cytometric MRD investigations (at least 4 measurements/patient: at day 30, between days 31–100, 101–180, and 181–365 after SCT) were performed measuring the following CLL phenotype: CD19posCD5posCD20dimCD79bneg. Therefore, a 4-color-approach (FACSCalibur, until 2006) or an 8-color-approach then in combination with T- and NK cell antigens (FACSCanto) was performed. A patient was defined as MRD negative if less than 0.05% CLL cells were detectable or as relapse if more than 0.05% CLL cells were again redetectable in at least two successive investigations. For assessment of progression-free survival (PFS), clinical progress was defined according to the NCI criteria. Results: The median follow up time after SCT was 536 days (range: 44d –1758d). Considering all 58 transplanted patients the probability of one-year overall survival (OS) including the 95% confidence interval was 83% ± 10% (2-year OS: 76%±12%) and of one-year PFS 74% ± 12% (2-year PFS: 50%±16%). In the majority of cases flow cytometric MRD negativity was achieved within the first year post SCT with a cumulative incidence of 36%±13% at day 100 and of 73%±12% at one year, respectively. Only two additional patients became MRD negative within the second year post SCT. Patients who achieved MRD negativity until day 365 showed a significantly better 2-year OS compared to the MRD positive group (96%±7% vs. 56%±49%; p=0.009). Remarkably, the 2-year PFS of patients achieving flow cytometric MRD negativity until day 365 was also significantly better than in the MRD positive cases (83%±16% vs. 0%, 3-year: 75%±20% vs. 0%; p=0.002). Of note, early flow cytometric MRD negativity until day 100 was not informative concerning OS or PFS at one year (88%±13% vs. 79%±15% and 77%±18% vs. 72%±18%). The flow cytometric MRD status was one trigger to speed the taper of cyclosporine or to give DLI. Interestingly, this kind of immunomodulation resulted in flow cytometric MRD negativity in eight out of nine patients after a median of 130 days. The probability of relapse in the investigated patient cohort was 15%±10% after 1 year and 31%±14% after 2 years. Thus, 8/14 patients showed a clinical relapse in parallel with flow cytometric MRD positivity. Two patients featured an isolated flow relapse with MRD at two successive investigations. Four patients showed a mere nodal relapse, all but one occurring within the first year post SCT. Conclusion: In summary, the present flow cytometric MRD study in B-CLL patients elucidates the dynamics of remission induction and relapse in the first year post SCT. In the majority of patients MRD is eradicated between day +100 and day +365 which is the time interval when chronic GvHD occurs in most cases. Therefore, close monitoring of MRD status in the first year after SCT is necessary. Once patients are flow cytometrically MRD negative at day 365, they seem to have a high probability of long term survival. Disclosures: Schetelig: Schering-Bayer: Research Funding.

Description: Gemtuzumab ozogamicin (GO) has been successfully used in older patients with relapsed CD33+ acute myeloid leukemia. Retrospective analyses suggest that the use of GO within a few months before or after hematopoietic stem cell transplantation (HCT) is associated with a increased risk for sinusoidal obstruction syndrome (SOS) or veno-occlusive liver disease (VOD). Ongoing studies investigate the use of GO in the induction and post-remission therapy of AML patients. We hypothesized that GO might be safe and effective as part of a reduced intensity conditioning regimen containing fludarabine and total-body irradiation (TBI) Fifteen patients relapsing after conventional induction chemotherapy (n=9) or after previous transplantation (autologous n=3; allogeneic n=3) were included so far. The last twelve patients were treated within an ongoing phase II trial. The preparative regimen contained 6 mg/m2 and 3 mg/m2 GO on day −21 and day −14 before allogeneic transplantation. Patients who responded to GO treatment and were below the age of 60 (n=8) received fludarabine 120 mg/m2 and 800 cGy TBI (n=6) during GO-induced aplasia followed by allogeneic HCT. Patients older than 60 years or those with relapsing AML

Description: Megakaryocytes, as precursor cells of platelets, comprise a cell population crucial for the maintenance of adult hematopoiesis. Since platelets are anuclear cellular fragments, the platelet transcriptome as well as the proteome are largely determined by the megakaryocyte. Calcium signaling in response to agonists such as collagen or thrombin has a central function in platelet activation and therefore likely represents an important signaling pathway in megakaryocytes as well. Indeed, megakaryocytes show considerable elevation of intracellular calcium levels in response to selected platelet agonists, but the downstream effector molecules of calcium signaling in these cells or the transcriptional responses induced are unknown. We here establish calcineurin and the NFAT (Nuclear Factor of Activated T cells) family of transcription factors as components of a calcium-induced signaling cascade in megakaryocytes. In resting megakaryocytes, NFAT is cytoplasmic and inactive, but can be activated by fibrillar collagen type 1, a physiological agonist of platelets and megakaryocytes known to induce a sustained increase in intracellular calcium levels in these cells. In contrast, treatment with SDF-1a, thrombopoietin, or VEGF remained without noticeable effect, presumably because of the short duration of the calcium transients induced by these agents. Collagen-induced NFAT activation in megakaryocytes requires dephosphorylation by calcineurin and is completely sensitive to the calcineurin inhibitor cyclosporin A. Activation of NFAT by collagen was paralleled by the induction of the expression of Down Syndrome Critical Region I (DSCR1) and Fas Ligand (FasL), two genes recently identified as NFAT targets in megakaryocytes. Collagen-induced expression of DSCR1 and FasL occurred in a calcineurin- and NFAT-dependent manner, as it was blocked by both cyclosporin A as well as the specific peptide inhibitor of NFAT, VIVIT. These experiments show that the calcineurin pathway is a target for selected physiological ligands capable of inducing sustained calcium mobilization in megakaryocytes and regulates megakaryocyte gene expression in a cyclosporin A- and NFAT-dependent manner.

Description: Besides graft versus host disease (GVHD), disease relapse is one of the major challenges in the care of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) undergoing allogeneic peripheral blood stem cell transplantation (PBSCT). However, we and others have shown that relapse can be predicted in case of CD34-expression on the malignant clone by a sensitive chimerism analysis in sorted CD34+ peripheral blood cells. If the percentage of donor cells in this compartment drops below 80%, leukemia relapse is inevitable within 4–8 weeks in the absence of clinical interventions like immediate cessation of immunosuppressive drugs or the administration of donor lymphocyte infusions. However, both approaches often result in clinically significant GVHD. Therefore, new strategies are warranted in order to treat imminent relapse in MDS or AML patients. We report results of an ongoing phase II clinical trial evaluating the efficacy of 5-azacitidine (5-aza) to prevent hematological relapse in patients with CD34+ AML or MDS and a decreasing CD34-donor chimerism after allogeneic PBSCT. Therefore, a total of 23 patients with CD34+ MDS (n=3) or AML (n=20) were prospectively screened on day 56, 84, 112, 140, 184, 365 and at later time points after PBSCT for a decreasing chimerism of donor CD34+ cells in the peripheral blood. In case of imminent relapse, defined as a drop of donor CD34+ cells below 80%, 5-aza was given at a dose of 75mg/m2/d s.c. day 1–7. A total of 4 cycles every 28 days were allowed in the absence of hematological relapse or toxicity. A median of 226 days after PBSCT, 9 out of 23 patients screened entered the treatment phase of the study with a median of 31% (range 0–53%) CD34+ donors cells in the peripheral blood (PB). However, a complete overall PB donor chimerism and less than 5% marrow blasts were documented in all patients before 5-aza treatment. Reversible neutropenia and thrombocytopenia grade 3/4 occurred in 50% of the patients. Only three patients still had immunosuppression prior 5-aza, which in two of them was slowly tapered during the period of 5-aza administration. With a median follow-up of 186 days after starting 5-aza all nine patients are eligible for response evaluation. Of these, CD34-chimerism was reverted to complete donor type (〉90%) in 6 (66%) patients. Two patients showed a further decrease of donor CD34+ cells and relapsed shortly after having completed the 1st or the 4th cycle of 5-aza, respectively. One patient showed an increase of CD34-chimerism after two cycles, however, died from non-relapse mortality. No hematological relapse occurred in the responders and in the screening cohort without decreasing CD34+ chimerism. Two patients (one without immunosuppression) developed limited cGVHD during 5-aza treatment. Preemptive treatment of minimal residual disease defined by decreasing donor CD34+ subset chimerism with 5-aza seems to be a potent strategy to prevent hematological relapse of CD34+ myeloid malignancies after allogeneic PBSCT.

Description: NFAT (Nuclear Factor of Activated T cells) is a family of calcium-induced, calcineurin-dependent transcription factors, well characterized as central regulators of inducible gene expression in T lymphocytes but now known to function also in several other cell types in various adaptation and differentiation processes. Activation of NFAT by the phosphatase calcineurin is counteracted by several inhibitory kinases and can be completely blocked by the immunosuppressant Cyclosporin A. The Down syndrome critical region 1 (DSCR1; also termed CSP1, MCIP1 or RCAN1) gene belongs to the calcipressin family of endogenous calcineurin inhibitors and is expressed in several isoforms, one of which (isoform C, coded by exons 4–7) has been described to be a transcriptional target for NFAT in striated muscle, endothelial, and neural cells. The DSCR1 gene is located within the Down syndrome critical region of human chromosome 21 and is, together with 200–300 other genes, overexpressed about 1.5-fold in patients with Down syndrome (DS). Previously, dysregulation of NFAT signaling by overexpression of DSCR1 has been implicated in causing various of the pathophysiological features observed in DS patients. Children with DS also suffer from an about 500-fold increased incidence of acute megakaryocytic leukemia; the respective roles of NFAT or DSCR1 in megakaryocytes of either normal individuals or those with DS, however, has not yet been established. Here we show that DSCR1 is upregulated during megakaryocytic differentiation in a lineage-specific manner, and in mature megakaryocytes is further strongly induced by calcineurin stimulation. DSCR1 expression in megakaryocytes is regulated by NFAT, since overexpression of NFATc2 enhances, while overexpression of the specific inhibitor of NFAT activation, VIVIT, suppresses expression of the gene. We further demonstrate that DSCR1 does not only represent an NFAT target in megakaryocytes, but itself acts an inhibitor of NFAT signaling in these cells. Overexpression of DSCR1 in CMK cells as well as in primary megakaryocytes by retroviral transduction profoundly suppressed ionomycin-induced dephosphorylation and nuclear translocation of NFATc2, as well as transactivation of an NFAT-dependent promoter construct. Finally, overexpression of DSCR1 in megakaryocytes markedly downregulated both the constitutive and induced expression of Fas Ligand, a pro-apoptotic gene recently established as a NFAT target in megakaryocytes. Together, these results suggest that DSCR1 acts as an NFAT-induced NFAT inhibitor in megakaryocytes and, when overexpressed, interferes with the expression of NFAT-dependent megakaryocytic genes.

Description: Abstract 3351 Poster Board III-239 Objectives: The majority of patients with chronic lymphocytic leukemia (CLL) who receive allogeneic hematopoietic cell transplantation (HCT) have fludarabine-refractory disease. The most active single agent in this disease stage is alemtuzumab. Alemtuzumab has a long half-life and induces profound T-cell depletion (TCD). Since TCD may mitigate graft-versus leukemia effects we evaluated „pre-conditioning“ with alemtuzumab followed by a washout period in order to minimize in vivo T-cell depletion of the graft in a phase II study (NCT 00337519). Methods: Patients received cytoreductive treatment with 3 × 30 mg alemtuzumab weekly prior to HCT. The scheduled interval between last dose of alemtuzumab and HCT was increased from two weeks to one month during the study. The antibody level at the day of HCT was measured with an ELISA with a lower limit of detection of 31.25 ng/mL (BioAnaLab lim., Oxford, UK). The conditioning regimen contained fludarabine (150 mg/m2) and busulfan (8 mg/kg). Cyclosporine (CSA) and methotrexate (MTX) were applied as GVHD-prophylaxis. Medically fit patients with relapsed CLL were elible. Results: 62 patients with a median age of 57 years were included between April, 2004 and October, 2008. A median of 3 prior regimens had been given. 55% of the patients had fludarabine-resistant disease. Two patients failed to reach HCT due to progressive disease during alemtuzumab therapy. Donors were matched siblings for 26 and matched unrelated donors for 34 patients. The median level of alemtuzumab in peripheral blood after a washout period of two weeks was 62 ng/mL (interquartile range, 45 to 196 ng/mL; minimum below the limit of detection; maximum 490 ng/mL) compared to a median level below the limit of detection after a delay of four weeks (interquartile range, between the limit of detection and 77 ng/mL, maximum 256 ng/mL) (p=0.005). Despite one month time between the last dose of alemtuzumab and HCT 4 out of 30 patients (13%) had alemtuzumab levels greater than 200 ng/mL. No primary or secondary graft failure occurred. A linear relationship between the alemtuzumab level at HCT and the time to complete CD4-T-cell chimerism (TCC) was observed (p=0.003). At day +100 a CD4 positive T-cell-chimerism (TCC) 〉95% had been achieved by 84% of patients with alemtuzumab levels 200 ng/mL (p=0.006). All patients had a complete neutrophil-chimerism at day +100. After early taper of immunosuppression (N=2) or the application of donor lymphocyte infusions in incremental doses (N=5) mixed TCC has been converted to complete TCC in all patients. The median follow-up is 17 months (1 to 61 months). Day +100 non-relapse mortality was 2%. At two years non-relapse mortality and relapse incidence were 21% and 29%, respectively. Two-year overall survival and progression-free survival were 67% (95% CI, 51% to 83%) and 50% (95% CI, 31% to 69%). Conclusions: In patients who received alemtuzumab prior to HCT, residual drug levels may interfere with T-cell engraftment. Lineage specific T-cell chimerism should therefore be assessed prospectively in this group of patients. Persistent mixed T-cell chimerism can be converted by an early taper of immunosuppression and incremental doses of donor lymphocyte infusions. Disclosures: Schetelig: Bayer Schering: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.