Effective and Successful Quantification of Leukemia-Specific Immune Cells in AML Patients' Blood or Culture, Focusing on Intracellular Cytokine and Degranulation Assays.

Novel (immune) therapies are needed to stabilize remissions or the disease in AML. Leukemia derived dendritic cells (DCleu) can be generated ex vivo from AML patients' blasts in whole blood using approved drugs (GM-CSF and PGE-1 (Kit M)). After T cell enriched, mixed lymphocyte culture (MLC) with Kit M pretreated (vs. untreated WB), anti-leukemically directed immune cells of the adaptive and innate immune systems were already shown to be significantly increased. We evaluated (1) the use of leukemia-specific assays [intracellular cytokine production of INFy, TNFa (INCYT), and degranulation detected by CD107a (DEG)] for a detailed quantification of leukemia-specific cells and (2), in addition, the correlation with functional cytotoxicity and patients' clinical data in Kit M-treated vs. not pretreated settings. We collected whole blood (WB) samples from 26 AML patients at first diagnosis, during persisting disease, or at relapse after allogeneic stem cell transplantation (SCT), and from 18 healthy volunteers. WB samples were treated with or without Kit M to generate DC/DCleu. After MLC with Kit M-treated vs. untreated WB antigen-specific/anti-leukemic effects were assessed through INCYT, DEG, and a cytotoxicity fluorolysis assay. The quantification of cell subtypes was performed via flow cytometry. Our study showed: (1) low frequencies of leukemia-specific cells (subtypes) detectable in AML patients' blood. (2) Significantly higher frequencies of (mature) DCleu generable without induction of blast proliferation in Kit M-treated vs. untreated samples. (3) Significant increase in frequencies of immunoreactive cells (e.g., non-naive T cells, Tprol) as well as in INCYT/DEG ASSAYS leukemia-specific adaptive-(e.g., B, T(memory)) or innate immune cells (e.g., NK, CIK) after MLC with Kit M-treated vs. untreated WB. The results of the intracellular production of INFy and TNFa were comparable. The cytotoxicity fluorolysis assay revealed significantly enhanced blast lysis in Kit M-treated vs. untreated WB. Significant correlations could be shown between induced leukemia-specific cells from several lines and improved blast lysis. We successfully detected and quantified immunoreactive cells at a single-cell level using the functional assays (DEG, INCYT, and CTX). We could quantify leukemia-specific subtypes in uncultured WB as well as after MLC and evaluate the impact of Kit M pretreated (DC/DCleu-containing) WB on the provision of leukemia-specific immune cells. Kit M pretreatment (vs. no pretreatment) was shown to significantly increase leukemia-specific IFNy and TNFa producing, degranulating cells and to improve blast-cytotoxicity after MLC. In vivo treatment of AML patients with Kit M may lead to anti-leukemic effects and contribute to stabilizing the disease or remissions. INCYT and DEG assays qualify to quantify potentially leukemia-specific cells on a single cell level and to predict the clinical course of patients under treatment.

Dendritic Cell-Triggered Immune Activation Goes along with Provision of (Leukemia-Specific) Integrin Beta 7-Expressing Immune Cells and Improved Antileukemic Processes.

Integrin beta 7 (ß7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell-cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific ß7 expression in immune cells after T-cell-enriched mixed lymphocyte culture-finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients' whole blood (WB) was treated with Kit-M (granulocyte-macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DCleu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DCleu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of ß7-expressing T-cells, degranulating and intracellular cytokine-producing ß7-expressing immune cells and, in patients' samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of ß7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) ß7-expressing immune cells. Furthermore, ß7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies.

Midostaurin added to chemotherapy and continued single-agent maintenance therapy in acute myeloid leukemia with FLT3-ITD.

Patients with acute myeloid leukemia (AML) and a FLT3 internal tandem duplication (ITD) have poor outcomes to current treatment. A phase 2 hypothesis-generating trial was conducted to determine whether the addition of the multitargeted kinase inhibitor midostaurin to intensive chemotherapy followed by allogeneic hematopoietic cell transplantation (alloHCT) and single-agent maintenance therapy of 12 months is feasible and favorably influences event-free survival (EFS) compared with historical controls. Patients 18 to 70 years of age with newly diagnosed AML and centrally confirmed FLT3-ITD were eligible: 284 patients were treated, including 198 younger (18-60 years) and 86 older (61-70 years) patients. Complete remission (CR) rate, including CR with incomplete hematological recovery (CRi) after induction therapy, was 76.4% (younger, 75.8%; older, 77.9%). The majority of patients in CR/CRi proceeded to alloHCT (72.4%). Maintenance therapy was started in 97 patients (34%): 75 after alloHCT and 22 after consolidation with high-dose cytarabine (HiDAC). Median time receiving maintenance therapy was 9 months after alloHCT and 10.5 months after HiDAC; premature termination was mainly a result of nonrelapse causes (gastrointestinal toxicity and infections). EFS and overall survival at 2 years were 39% (95% confidence interval [CI], 33%-47%) and 34% (95% CI, 24%-47%) and 53% (95% CI, 46%-61%) and 46% (95% CI, 35%-59%) in younger and older patients, respectively. EFS was evaluated in comparison with 415 historical controls treated within 5 prospective trials. Propensity score-weighted analysis revealed a significant improvement of EFS by midostaurin (hazard ratio [HR], 0.58; 95% CI, 0.48-0.70; P < .001) overall and in older patients (HR, 0.42; 95% CI, 0.29-0.61). The study was registered at www.clinicaltrials.gov as #NCT01477606.

Smokers with chronic myeloid leukemia are at a higher risk of disease progression and premature death.

BACKGROUND: Smoking is suspected to not only be a risk factor for chronic myeloid leukemia but an adverse prognostic factor for the disease as well. The objective of the current study was to investigate the impact of smoking on survival and progression to advanced phases of disease. METHODS: Based on the data of the German CML Study IV, the authors analyzed the effect of smoking using a multivariate Cox model with the addition of the European Treatment and Outcome Study (EUTOS) long-term survival score variables of age, spleen size, thrombocytes, and peripheral blasts as well as sex, comorbidities, and type of treatment center. RESULTS: The 8-year survival probability was 87% for a nonsmoking patient and 83% for a patient who smoked. The authors noted a 2.08-times higher risk of death for smokers in comparison with nonsmokers and a 2.11-times higher cause-specific hazard of disease progression. An interaction between smoking and age was found in the model for survival. No significant difference with regard to molecular response was observed. CONCLUSIONS: Even when considering differences in socioeconomic status and lifestyle between patients who smoke and nonsmokers, the current analysis demonstrated that smoking also might affect disease biology. The results of the current study indicate that patients with chronic myeloid leukemia, in particular those aged <60 years, should be encouraged to quit smoking. Cancer 2017;123:2467-71. © 2017 American Cancer Society.

Impact of comorbidities on overall survival in patients with chronic myeloid leukemia: results of the randomized CML study IV.

We studied the influence of comorbidities on remission rate and overall survival (OS) in patients with chronic myeloid leukemia (CML). Participants of the CML Study IV, a randomized 5-arm trial designed to optimize imatinib therapy, were analyzed for comorbidities at diagnosis using the Charlson Comorbidity Index (CCI); 511 indexed comorbidities were reported in 1519 CML patients. Age was an additional risk factor in 863 patients. Resulting CCI scores were as follows: CCI 2, n = 589; CCI 3 or 4, n = 599; CCI 5 or 6, n = 229; and CCI ≥ 7, n = 102. No differences in cumulative incidences of accelerated phase, blast crisis, or remission rates were observed between patients in the different CCI groups. Higher CCI was significantly associated with lower OS probabilities. The 8-year OS probabilities were 93.6%, 89.4%, 77.6%, and 46.4% for patients with CCI 2, 3 to 4, 5 to 6, and ≥7, respectively. In multivariate analysis, CCI was the most powerful predictor of OS, which was still valid after removal of its age-related components. Comorbidities have no impact on treatment success but do have a negative effect on OS, indicating that survival of patients with CML is determined more by comorbidities than by CML itself. OS may therefore be inappropriate as an outcome measure for specific CML treatments. The trial was registered at www.clinicaltrials.gov as #NCT00055874.

Platelet recovery and survival measured in patients by quantitative polymerase chain reaction of mitochondrial DNA.

BACKGROUND: Mitochondrial (mt) DNA markers have been identified as potential targets for the quantification of endogenous and allogeneic platelets (PLTs) in the blood of individuals who received transfusions. Our goal was to develop a routine polymerase chain reaction (PCR) assay for ex vivo monitoring of PLT survival in patients after transfusion. STUDY DESIGN AND METHODS: Targets were selected for real-time (RT)-PCR of mt DNA based on the frequency distribution of nucleotide polymorphisms and assay sensitivity in vitro. The assays were then evaluated with ex vivo samples to measure PLT survival and recovery of therapeutic doses of apheresis PLTs in hematooncologic patients with thrombocytopenia. RESULTS: Nucleotides in two positions (73/310 hypervariable region [HVR] 2) and three positions (295 HVR 2, 16069/16311 HVR 1) had allele frequencies of approximately 0.5 and 0.85, respectively, in a population of 960 Caucasian PLT donors. They provided targets for sensitive assays detecting at least 1 × 10(3) PLTs per whole blood sample with adequate reproducibility (interassay coefficient of variation <4.0%). Transfusions of single-donor PLT concentrates in patients with thrombocytopenia (n = 30) were monitored with these markers. The mean 24-hour corrected count increment was 8.3 and the mean calculated survival time was 3.3 days. Results for a second marker were available for 13 transfusions. The survival time values derived from both markers for the same transfusion were almost identical (linear regression: r(2) = 0.957, slope = 0.87). CONCLUSION: This RT-PCR method detects mt DNA polymorphisms in Caucasians for a highly sensitive and reproducible quantification of endogenous and allogeneic PLT numbers in blood samples from transfused patients with thrombocytopenia.

Long-term clinical and molecular remissions in patients with mantle cell lymphoma following high-dose therapy and autologous stem cell transplantation.

Long-term clinical and molecular remissions in patients with mantle cell lymphoma (MCL) following high-dose therapy (HDT) and autologous stem cell transplantation (ASCT) have been evaluated in only a few studies. Thirty-six patients with MCL received ASCT in our institution (27 patients undergoing first-line therapy, 8 patients undergoing second-line therapy, and 1 patient undergoing third-line therapy). In the case of long-term remission (≥5 years; n = 8), peripheral blood was tested for minimal residual disease (MRD) by t(11; 14) polymerase chain reaction (PCR) and immunoglobulin heavy-chain (IGH) PCR at the last follow-up. Ten-year overall survival (OS), progression-free survival (PFS), and freedom from progression (FFP) after first-line ASCT were 42 %, 43 %, and 54 %; after second-line ASCT, these were all 0 %. Four-year OS, PFS, and FFP for the first-line cohort were 75 %, 48 %, and 61 %, respectively. Four-year OS, PFS, and FFP after second-line ASCT were 55 %, 30 %, and 30 %, respectively. Treatment-related mortality (3 months after ASCT) was 0 %. The only prognostic factor for OS, PFS, and FFP was treatment line (p = 0.011, p = 0.046, and p = 0.023, respectively). No relapses occurred after 5 years following ASCT. So far, eight patients developed sustained long-term clinical and molecular complete remissions of up to 14.6 years following ASCT in the first treatment line. Sustained long-term clinical and molecular remissions can be achieved following ASCT in the first treatment line and apparently less frequent in the second treatment line.

Therapeutic drug monitoring-guided treatment versus standard dosing of voriconazole for invasive aspergillosis in haematological patients: a multicentre, prospective, cluster randomised, crossover clinical trial.

OBJECTIVES: Voriconazole therapeutic drug monitoring (TDM) is recommended based on retrospective data and limited prospective studies. This study aimed to investigate whether TDM-guided voriconazole treatment is superior to standard treatment for invasive aspergillosis. METHODS: A multicentre (n = 10), prospective, cluster randomised, crossover clinical trial was performed in haematological patients aged ≥18 years treated with voriconazole. All patients received standard voriconazole dose at the start of treatment. Blood/serum/plasma was periodically collected after treatment initiation of voriconazole and repeated during treatment in both groups. The TDM group had measured voriconazole concentrations reported back, with dose adjustments made as appropriate, while the non-TDM group had voriconazole concentrations measured only after study completion. The composite primary endpoint included response to treatment and voriconazole treatment discontinuation due to an adverse drug reaction related to voriconazole within 28 days after treatment initiation. RESULTS: In total, 189 patients were enrolled in the study. For the composite primary endpoint, 74 patients were included in the non-TDM group and 68 patients in the TDM group. Here, no significant difference was found between both groups (P = 0.678). However, more trough concentrations were found within the generally accepted range of 1-6 mg/L for the TDM group (74.0%) compared with the non-TDM group (64.0%) (P < 0.001). CONCLUSIONS: In this trial, TDM-guided dosing of voriconazole did not show improved treatment outcome compared with standard dosing. We believe that these findings should open up the discussion for an approach to voriconazole TDM that includes drug exposure, pathogen susceptibility and host defence. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov registration no. NCT00893555.

Prediction of Bone Marrow Biopsy Results From MRI in Multiple Myeloma Patients Using Deep Learning and Radiomics.

OBJECTIVES: In multiple myeloma and its precursor stages, plasma cell infiltration (PCI) and cytogenetic aberrations are important for staging, risk stratification, and response assessment. However, invasive bone marrow (BM) biopsies cannot be performed frequently and multifocally to assess the spatially heterogenous tumor tissue. Therefore, the goal of this study was to establish an automated framework to predict local BM biopsy results from magnetic resonance imaging (MRI). MATERIALS AND METHODS: This retrospective multicentric study used data from center 1 for algorithm training and internal testing, and data from center 2 to 8 for external testing. An nnU-Net was trained for automated segmentation of pelvic BM from T1-weighted whole-body MRI. Radiomics features were extracted from these segmentations, and random forest models were trained to predict PCI and the presence or absence of cytogenetic aberrations. Pearson correlation coefficient and the area under the receiver operating characteristic were used to evaluate the prediction performance for PCI and cytogenetic aberrations, respectively. RESULTS: A total of 672 MRIs from 512 patients (median age, 61 years; interquartile range, 53-67 years; 307 men) from 8 centers and 370 corresponding BM biopsies were included. The predicted PCI from the best model was significantly correlated ( P ≤ 0.01) to the actual PCI from biopsy in all internal and external test sets (internal test set: r = 0.71 [0.51, 0.83]; center 2, high-quality test set: r = 0.45 [0.12, 0.69]; center 2, other test set: r = 0.30 [0.07, 0.49]; multicenter test set: r = 0.57 [0.30, 0.76]). The areas under the receiver operating characteristic of the prediction models for the different cytogenetic aberrations ranged from 0.57 to 0.76 for the internal test set, but no model generalized well to all 3 external test sets. CONCLUSIONS: The automated image analysis framework established in this study allows for noninvasive prediction of a surrogate parameter for PCI, which is significantly correlated to the actual PCI from BM biopsy.

Leukemia derived dendritic cell (DCleu) mediated immune response goes along with reduced (leukemia-specific) regulatory T-cells.

The blastmodulatory Kit-M, composed of granulocyte-macrophage colony-stimulating-factor (GM-CSF) and Prostaglandin E1 (PGE1), is known to convert myeloid leukaemic blasts (from AML patients) into leukaemia derived dendritic cells (DCleu), which activate immunoreactive cells to gain antileukemic/leukaemia-specific activity. In this study we had a special focus on the influence of Kit-M treated, DC/DCleu containing patients'whole blood (WB, n = 16) on the provision of immunosuppressive regulatory T-cells. We could confirm that Kit-M significantly increased frequencies of (mature) dendritic cells (DC) and DCleu from leukemic whole blood (WB) without induction of blast proliferation. After mixed lymphocyte culture (MLC) with patients' T-cells we confirmed that DCleu mediated leukemia-specific responses- going along with activated and leukemia-specific T- and NK-cells in an intracellular cytokine staining assay (ICS) and a degranulation assay (Deg)- resulted in an increased anti-leukemic cytotoxicity (Cytotoxicity Fluorolysis Assay = CTX). We could demonstrate that (leukemia-specific) CD4+ and CD8+ regulatory T-cell population (Treg) decreased significantly after MLC compared to controls. We found significant positive correlations of leukemia-specific CD3+CD4+ cells with frequencies of (mature) DCleu. Achieved anti-leukemic cytotoxicity correlated significantly positive with leukemia-specific CD3+CD8+ cells and significantly negatively with (leukemia-specific) Treg. In summary we demonstrate that immunesuppressive (leukemia-specific) regulatory T-cells are significantly downregulated after Kit-M triggered MLC- going along with a (reinstalled) antileukemic reactivity of the immune system (as demonstrated with functional assays ICS, Deg, CTX).

Expression of Surface-associated 82 kDa proMMP-9 in Lymphatic Leukemia Blast Cells Differentially Correlates With Prognosis.

BACKGROUND/AIM: Matrix metalloproteinases (MMPs) degrade extracellular matrix and process regulatory proteins. Recently, a membrane-bound 82kDa variant of proMMP-9 identified on myeloid blasts was shown to be associated with prognosis. PATIENTS AND METHODS: To investigate the role of 82kDa proMMP-9 with acute lymphoblastic leukemia (ALL) and chronic lymphoid leukemia (CLL), we performed flow-cytometry analysis of expression on ALL blasts (n=18) and CLL lymphocytes (n=21) from blood and correlated data with clinical parameters. RESULTS: In ALL, mature B-linear blasts expressed higher levels of 82kDa proMMP-9 compared to T-linear blasts. Elevated levels of 82kDa proMMP-9 were found in elderly patients and at patients with relapse. No correlation was observed on blood cells and extramedullary disease. In CLL, the 82kDa proMMP-9 expression did not correlate with any of the clinical parameters. CONCLUSION: Our findings suggest that higher levels of 82kDa proMMP-9 expression on blast cells may correlate with a more unfavorable ALL-subtype. Further studies are required to clarify the prognostic role of the 82kDa pro-MMP-9 expression.

Role of Interferon (IFN)α in "Cocktails" for the Generation of (Leukemia-derived) Dendritic Cells (DCleu) From Blasts in Blood From Patients (pts) With Acute Myeloid Leukemia (AML) and the Induction of Antileukemic Reactions.

Strategies to stabilize remissions by specific elimination of residual acute myeloid leukemia (AML) blasts are needed. Leukemia-derived dendritic cell (DCleu/DC) generated from myeloid blasts improve antileukemic T-cell reactivity and install T-cell memory. Interferon (IFN)α-DC methods produce DCleu from chronic myeloid leukemia-patients (pts') blood. Various INFα-containing versus other DC methods were studied to produce DCleu (evaluated by flowcytometry) from AML-pts' blast-containing mononuclear (MNC) or whole blood (WB). After DCleu/DC stimulation in mixed lymphocyte cultures, T cells' potential to gain antileukemic cytotoxicity was studied and correlated with different DC methods and DCleu/DC counts. (1) Generation of DCleu/DC: (a) "IFN-GIT" [containing granulocyte macrophage-colony stimulating factor (GM-CSF)+IFNα+ tumor necrosis factor (TNF)-α] produced DC successfully (≥10% DC, ≥5% DCleu/cells) from AML-MNC (WB) in 54 (56%), "MCM-Mimic" in 76 (75%), "Picibanil" in 83 (64%), and "Calcium-ionophore" in 42 (67%) of cases. Proportions of DC subtypes in MNC (WB) were comparable with all DC methods, (b) IFNα combinations containing only GM-CSF+IFNα or only IFNα showed low efficiency to produce DCleu/DC from MNC (WB) compared with "IFN-GIT." (2) Antileukemic functionality: DCleu/DC-stimulated T cells showed improved leukemia cytotoxicity compared with blast cells or unstimulated T cells. The highest blast proliferation (=insufficient T cells) was seen with "IFN-GIT" DC-stimulated T cells. Probability to respond to immunotherapy or to obtain blast lysis of DC-stimulated T cells correlated with high proportions of DCleu/DC after DC culture, independent of DC-generating methods. (3) Cytokine release profiles: levels of interleukin-6, IFN-γ, and interleukin-2 were significantly lower in DC culture supernatants (from MNC/WB) with "IFN-GIT" compared with "MCM," "Pici," and "Ca" DC supernatants. Our data show that (1) WB culture simulates AML-pts' in vivo situation, (2) DC generation is possible from AML-MNC (WB) with IFNα-containing and other DC methods, (3) successful IFNα-DC generation needs GM-CSF+IFNα+TNF-α (IFN-GIT); however, "IFN-GIT" produces less DCleu/DC compared with other (non-IFNα) DC methods, (4) T cells stimulated with "IFN-GIT"-produced DCleu/DC yielded comparable antileukemic cytotoxicity; however, in cases without achieved blast lysis, an increased blast proliferation was observed.

Platelet engraftment after allogenic stem cell transplantation is monitored by digital polymerase chain reaction without interference by platelet support.

Platelet engraftment after allogeneic hematopoietic stem cell transplantation is conventionally monitored by daily platelet counts. Platelet transfusions are frequently required and obscure the detection of platelet engraftment. Digital polymerase chain reaction (ddPCR) of mitochondrial DNA isolated from platelets reliably quantifies circulating platelets derived from the stem cell graft and allows us to distinguish them from transfused single-donor apheresis platelets. In a feasibility study, consecutive daily peripheral blood samples from day 7 to day 20 after transplantation were analyzed by ddPCR in 22 patients after allogeneic transplantation. Platelet engraftment according to ddPCR was defined as the third of at least 3 consecutive days of increasing levels exceeding 1000/µL endogenous platelets. Platelet counts were also assessed according to the engraftment cri`teria of the Center for International Blood & Marrow Transplant Research (CIBMTR) and the European Society for Blood and Marrow Transplantation (EBMT). Out of the 22 patients, five did not achieve platelet engraftment within 20 days by any of the predefined criteria. A subgroup of nine patients did show platelet engraftment by all three definitions. In five patients, engraftment was detectable according to ddPCR and EBMT, whereas in three patients, platelet engraftment within 20 days was only confirmed by ddPCR. The detailed findings suggest that the day of platelet engraftment according to the EBMT criteria closely reflected the ddPCR detection of transplantation-derived platelets. The results from this feasibility study demonstrate that ddPCR offers a sensitive approach to detect platelet engraftment reliably and without interference from the individual noise of platelet counts due to platelet transfusions.

Adding dasatinib to intensive treatment in core-binding factor acute myeloid leukemia-results of the AMLSG 11-08 trial.

In this phase Ib/IIa study (ClinicalTrials.gov Identifier: NCT00850382) of the German-Austrian AML Study Group (AMLSG) the multikinase inhibitor dasatinib was added to intensive induction and consolidation chemotherapy and administered as single agent for 1-year maintenance in first-line treatment of adult patients with core-binding factor (CBF) acute myeloid leukemia (AML). The primary combined end point in this study was safety and feasibility, and included the rates of early (ED) and hypoplastic (HD) deaths, pleural/pericardial effusion 3°/4° and liver toxicity 3°/4°, and the rate of refractory disease. Secondary end points were cumulative incidence of relapse (CIR) and death in complete remission (CID), and overall survival (OS). Eighty-nine pts [median age 49.5 years, range: 19-73 years; t(8;21), n = 37; inv (16), n = 52] were included. No unexpected excess in toxicity was observed. The rates of ED/HD and CR/CRi were 4.5% (4/89) and 94% (84/89), respectively. The 4-year estimated CIR, CID, and OS were 33.1% [95%-CI (confidence interval), 22.7-43.4%], 6.0% (95% CI, 0.9-11.2%), and 74.7% (95% CI, 66.1-84.5%), respectively. On the basis of the acceptable toxicity profile and favorable outcome in the AMLSG 11-08 trial, a confirmatory randomized phase III trial with dasatinib in adults with CBF-AML is ongoing (ClinicalTrials.gov Identifier: NCT02013648).

Cytokine Release Patterns in Mixed Lymphocyte Culture (MLC) of T-Cells with Dendritic Cells (DC) Generated from AML Blasts Contribute to Predict anti-Leukaemic T-Cell Reactions and Patients' Response to Immunotherapy.

To enlighten interactions between autologous, allogeneic or T-cells from patients after stem cell transplantation with leukaemia-derived-dendritic-cells containing dendritic cells or blast containing mononuclear cells (n = 21, respectively), we determined cytokine-concentrations (interleukin 2, 4, 6, 10, tumor-necrosis-factor-α, interferon-γ) in supernatants of mixed-lymphocyte-culture and in serum (n = 16) of 20 patients with acute myeloid leukaemia and three patients with myelodysplastic syndromes by cytometric-bead-assay. We correlated our data with lytic capabilities of stimulated T-cells in a fluorolysis-assay and clinical data: Dendritic-cell-/mononuclear-cell-stimulation of T-cells resulted in increased cytokine-levels in culture-medium compared to serum. There were no significant differences between cytokine-patterns of cases with/without lytic T-cell-activity, response to immunotherapy (stem cell transplantation/donor-lymphocyte-infusion) or graft-versus-host-disease. However, some predictive cytokine-cut-off-values for antileukaemic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease could be defined. Cytokine-profiles alone, without functional assays, are no useful tool to predict antileukaemic T-cell-function, although they can indicate lytic T-cell-activity, patients' response to immunotherapy and graft-versus-host-disease.

Kidney Na+,K(+)-ATPase is associated with moesin.

Na+,K(+)-ATPase is a ubiquitous plasmalemmal membrane protein essential for generation and maintenance of transmembrane Na+ and K+ gradients in virtually all animal cell types. Activity and polarized distribution of renal Na+,(+)-ATPase appears to depend on connection of ankyrin to the spectrin-based membrane cytoskeleton as well as on association with actin filaments. In a previous study we showed copurification and codistribution of renal Na+,K(+)-ATPase not only with ankyrin, spectrin and actin, but also with two further peripheral membrane proteins, pasin 1 and pasin 2. In this paper we show by sequence analysis through mass spectrometry as well as by immunoblotting that pasin 2 is identical to moesin, a member of the FERM (protein 4.1, ezrin, radixin, moesin) protein family, all members of which have been shown to serve as cytoskeletal adaptor molecules. Moreover, we show that recombinant full-length moesin as well as its FERM domain bind to Na+,K(+)-ATPase and that this binding can be inhibited by an antibody specific for the ATPase activity-containing cytoplasmic loop (domain 3) of the Na+,K(+)-ATPase alpha-subunit. This loop has been previously shown to be a site essential for ankyrin binding. These observations indicate that moesin might not only serve as direct linker molecule of Na+,K(+)-ATPase to actin filaments but also modify ankyrin binding at domain 3 of Na+,K(+)-ATPase in a way similar to protein 4.1 modifying the binding of ankyrin to the cytoplasmic domain of the erythrocyte anion exchanger (AE1).

[Gilbert disease and type I and II Crigler-Najjar syndrome due to mutations in the same UGT1A1 gene locus]. / Morbus Gilbert und Crigler-Najjar-Syndrom Typ I und II beruhen auf Mutationen im selben Genlocus UGT1A1.

BACKGROUND: Gilbert syndrome and the Crigler-Najjar syndromes Type I and II are disorders of bilirubin conjugation with consecutive indirect hyperbilirubinemia of different severity. Morbus Gilbert is a mild hyperbilirubinemia, which is only of significance in case of drug therapy or differential diagnosis. Crigler-Najjar syndrome II leads to a more serious kind of hyperbilirubinemia. In case of Crigler-Najjar syndrome I patients are suffering from a very severe hyperbilirubinemia, which often causes death during the first months of life. MOLECULAR GENETICS: The molecular defects of these three syndromes have been characterized during the last decade. They are caused by mutations in the UGT1A1 gene locus. This locus codes for the enzyme bilirubin uridine 5'-diphosphate-(UDP-) glucuronosyltransferase (UGT1A1). In the case of Gilbert syndrome two bases are inserted into the promoter of the gene. In Crigler-Najjar syndrome type I and II mutations lead to the exchange of amino acids, changes of the reading frame or to stop codons. CONCLUSION: All three forms of indirect hyperbilirubinemia are caused by mutations in the UGT1A1 gene locus, which codes for the enzyme UDP-glucuronosyltransferase.

[Systemic mastocytosis. Classification, symptoms, therapy]. / Systemische Mastozytose. Klassifikation, Symptome und Therapie.

BACKGROUND: Systemic mastocytoses are a group of diseases, which are characterized by accumulation and unusual growth of mast cells infiltrating two different organs or types of tissue. Two case reports are introduced. CLASSIFICATION: According to the new WHO classification of 2000, mastocytoses are separated into cutaneous and systemic mastocytoses. Systemic mastocytosis is subdivided into an indolent course with good prognosis and four subgroups with poor prognosis (systemic mastocytosis with associated clonal hematologic non-mast-cell disease, aggressive systemic mastocytosis, mast cell leukemia, and mast cell sarcoma). GENETICS: Systemic mastocytoses are clonal disorders of mast cells and their progenitor cells, which may show point mutations of the protooncogene c-kit. This gene codes for the stem cell receptor (CD117). THERAPY: Therapy of systemic mastocytosis depends on patient's symptoms. There is no known cure of the disease. Besides diet and avoidance of skin irritations, symptoms are treated with H(1)- or H(2)-blockers, steroids, leukotriene receptor antagonists, and PUVA therapy. If patients suffer from systemic reactions such as hypotension or syncope, epinephrine solution should be prescribed for emergency use.

Profiles of activation, differentiation-markers, or ß-integrins on T cells contribute to predict T cells' antileukemic responses after stimulation with leukemia-derived dendritic cells.

Stem cell transplantations and donor lymphocyte infusions are promising immunotherapies to cure acute myeloid leukemia (AML). Leukemia-derived dendritic cells are known to improve antileukemic functionality of T cells. We evaluated the composition and development of distinct T-cell subtypes in AML patients (n=12) compared with healthy probands (n=5) before and during stimulation with leukemia-derived dendritic cells-containing DC (DC) or blast-containing mononuclear cells (MNC) in 0-7 days mixed lymphocyte cultures (MLC) by flow cytometry. AML patients' T-cell subgroups were correlated with antileukemic functionality before and after DC/MNC stimulation by functional fluorolysis assays. (1) Unstimulated T cells from AML patients presented with significantly lower proportions of activated, Tcm, CD137, and ß-integrin T cells, and significantly higher proportions of Tnaive and Teff compared with healthy probands. (2) After 7 days of DC or MNC stimulation, T-cell profiles were characterized by (significantly) increased proportions of activated T cells with effector function and significantly decreased proportions of ß-integrin T cells. (3) Antileukemic cytotoxicity was achieved in 40% of T cells after MNC stimulation compared with 64% after DC stimulation. Antileukemic activity after DC stimulation but not after MNC stimulation correlated with higher proportions of Tcm and Tnaive before stimulation, as well as with significantly higher proportions of activated and ß-integrin T cells. Furthermore, cutoff values for defined T-cell activation/differentiation markers and ß-integrin T cells could be defined, allowing a prediction of antileukemic reactivity. We could demonstrate the potential of the composition of unstimulated/DC-stimulated T cells for the lysis of AML blasts. Especially, AML patients with high numbers of Tnaive and Tcm could benefit from DC stimulation; proportions of activated and ß-integrin T cells correlated with increased antileukemic functionality and could serve to predict T cells' reactivity during stimulation. Refined analyses in the context of responses to immunotherapies are required.