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Cytokines play a key role in regulation of normal and malignant hematopoiesis, angiogenesis, and inflammation. Serum levels of several cytokines are altered in patients with hematologic malignancies, and pretransplant cytokine levels seem to have a prognostic impact in patients treated with allogeneic stem cell transplantation. However, the cytokine system constitutes an interacting functional network, and it may therefore be more relevant to look at serum cytokine profiles rather than the serum levels of single cytokines in allotransplanted patients. We therefore investigated the pretransplantation serum levels of 35 cytokines in a group of 44 consecutive allogeneic stem cell transplantation patients, mainly with a primary diagnosis of acute leukemia. Serum samples were collected before the start of myeloablative conditioning therapy when all patients were in complete hematologic remission. Unsupervised hierarchical clustering analysis identified three major patient groups/subsets. These groups differed especially in the levels of hepatocyte growth factor and granulocyte-colony stimulating factor, and one of the groups was characterized by low early treatment-related morbidity and high levels of hepatocyte growth factor and granulocyte-colony stimulating factor. The degree of weight gain/fluid retention after conditioning therapy did not differ between the patient subsets, but fluid retention showed a significant correlation with pretransplantation serum levels of basic fibroblast growth factor. We conclude that the pretransplantation serum cytokine profile shows a considerable variation even between patients in complete hematologic remission and is associated with clinicopathologic features.

Several studies report that patients who are treated with selective serotonin reuptake inhibitors (SSRIs) for depression may have increased risk of bleeding, particularly from the gastrointestinal tract. This may be related to low intraplatelet serotonin concentrations. Several blood banks do not store platelets from donors using SSRIs for transfusion, although the possible effects of SSRIs on platelet storage are not documented. We conducted a case-control pilot study of apheresis platelet concentrates prepared from donors using SSRIs (n=8) and from donors without medication (n=10). The platelet concentrates were stored for 5 days. Light transmission aggregometry (LTA), thrombelastography (TEG), and flow cytometric analyses were preformed for in vitro measurements of platelet function. Platelet function and platelet serotonin content were investigated in whole blood and in platelet concentrates stored for up to 5 days. LTA, TEG, and flow cytometric analysis of glycoprotein expression did not reveal any significant differences between the two groups. All 18 platelet concentrates performed well according to the standards set for platelet quality in relation to transfusion. Blood donors using SSRIs had significantly lower platelet serotonin compared to blood donors without medication. The results from our pilot study indicate that platelets from donors using SSRIs may be suitable for transfusion after storage for 5 days, but further laboratory and clinical studies are necessary to confirm this.

Heat shock proteins (HSPs) are molecular chaperones that assist proteins in their folding to native structures. HSPs are regarded as possible therapeutic targets in acute myeloid leukaemia (AML). We used bioinformatical approaches to characterize the HSP profile in AML cells from 75 consecutive patients, in addition to the effect of the HSP90 inhibitor 17-DMAG. Patients harbouring a FLT3-internal tandem duplication (FLT3-ITD) were extensively overrepresented in the cluster with high HSP levels, indicating a strong dependence of HSPs in stabilizing FLT3-ITD encoded oncoproteins. FLT3 ligation further increased the levels of HSP90 and its co-chaperone HSP70. HSP90 inhibition had a stronger pro-apoptotic effect for AML cells with FLT3-ITD than for cells with wild-type FLT3, whereas the anti-proliferative effect of HSP90 inhibition was similar for the two patient subsets. HSP90 inhibition altered the constitutive cytokine release profile in an anti-angiogenic direction independent of FLT3 mutational status: (i) pro-angiogenic CXCL8, MMP-2 and MMP-9 showed a stronger decrease than anti-angiogenic CXCL9-11, (ii) the Tie-2 agonist Ang-1 showed a stronger decrease than the potentially antagonistic Ang-2, and (iii) VEGF and HGF levels were decreased. Finally, HSP90 inhibition counteracted the leukaemia-stimulating effect of endothelial cells. Our studies demonstrate that HSP90 inhibition mediates anti-leukaemic effects through both direct and indirect activity.

Polycythemia vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms (MPNs), which generally follow a benign and indolent clinical course. However, venous thromboses are common and constitute the main cause of morbidity and mortality. The discovery of the JAK2V617F mutation and other biomarkers has advanced our understanding of these diseases. There is a strong association between the presence of the JAK2V617F mutation and the development of thrombosis in ET. If venous thrombosis presents with unusual manifestations, the diagnosis of a MPN, such as PV or ET, should be part of the differentials. Treatment of venous thrombosis in MPN follows the same principle as in other patients with venous thrombosis, but careful attention to primary and secondary prophylaxis in addition to heparin-induced thrombocytopenia should be given. Cytoreductive therapy is indicated in high-risk subgroups of PV and ET patients, and alternative therapeutic agents have different effects on risk of venous thrombosis. New therapeutic approaches are emerging, and JAK2 inhibitors, histone deacetylase inhibitors and next-generation anticoagulants are in various stages of clinical development for the treatment of MPN, but their exact role in thrombosis prevention and treatment remains unclear.

Platelet concentrates contain soluble mediators derived from both platelets and contaminating leukocytes. During platelet transfusion these mediators are transferred, and transfusion-induced modulation of the cytokine network may then occur, possibly contributing to transfusion reactions, immunomodulation, or affecting residual leukemic cells. In this prospective observational study, we investigate the effect of platelet transfusion on the systemic levels of platelet-derived cytokines, chemokines and interleukins in an unselected group of acute leukaemia patients with severe chemotherapy-induced cytopenia. We investigated 31 platelet transfusions involving pre-storage, white blood cell-reduced, gamma-irradiated or pathogen-inactivated, photochemically-treated platelet concentrates received by 10 unselected patients. Peripheral blood plasma samples were collected before, immediately after, one hour, and 24 hours after the transfusions. Sampling from platelet concentrates was performed immediately before transfusion. A total of 31 soluble mediators were examined. Ten healthy controls matched for age and gender were included. Despite heterogeneity in patients and platelet concentrates, significantly increased plasma concentrations were detected for the platelet-derived mediators, platelet-derived growth factor, β-thromboglobulin, transforming growth factor-β (TGF-β), CCL5, and CXCL4, 1 hour and/or immediately after platelet transfusions. The plasma levels of vascular endothelial growth factor and soluble CD40 ligand were not altered by platelet transfusion. Certain interleukins (IL-1β, IL-2, IL-4, IL-6, IL-9, and IL-12), as well as interferon-γ showed a minor, transient decrease in systemic plasma levels during the first hour following transfusion. Platelet transfusions modulate the systemic cytokine network in acute leukaemia patients with severe, chemotherapy-induced cytopenia.

Protein and gene networks centred on the regulatory tumour suppressor proteins may be of crucial importance both in carcinogenesis and in the response to chemotherapy. Tumour suppressor protein p53 integrates intracellular data in stress responses, receiving signals and translating these into differential gene expression. Interpretation of the data integrated on p53 may therefore reveal the response to therapy in cancer. Proteomics offers more specific data - closer to "the real action" - than the hitherto more frequently used gene expression profiling. Integrated data analysis may reveal pathways disrupted at several regulatory levels. Ultimately, integrated data analysis may also contribute to finding key underlying cancer genes. We here proposes a Partial Least Squares Regression (PLSR)-based data integration strategy, which allows simultaneous analysis of proteomic data, gene expression data and classical clinical parameters. PLSR collapses multidimensional data into fewer relevant dimensions for data interpretation. PLSR can also aid identification of functionally important modules by also performing comparison to databases on known biological interactions. Further, PLSR allows meaningful visualization of complex datasets, aiding interpretation of the underlying biology. Extracting the true biological causal mechanisms from heterogeneous patient populations is the key to discovery of new therapeutic options in cancer.

Matrix metalloproteinases (MMPs) comprise a large family of zinc-dependent endopeptidases, which are best known for their ability to degrade essentially all components of the extracellular matrix (ECM). By breaking down ECM, MMPs may remove physical barriers, thus allowing cells to migrate and potentially invade other tissues. Recent evidence, however, shows that the proteolytic activities of MMPs also affect several fundamental physiological processes. Primary human acute myeloid leukemia (AML) cells often show constitutive release of several MMPs and chemokines, and there seems to be a crosstalk between the MMP system and the chemokine network. Firstly, the nuclear factor-κB (NF-κB) system represents a common regulator at the transcriptional level both for MMPs (e.g. MMP-1 and MMP-9) and for the constitutive release of several chemokines (CCL2-4/CXCL1/8) by primary human AML cells. Secondly, the crosstalk at the molecular level probably includes MMP-mediated structural alteration and activation of constitutively released chemokines involved in AML cell migration (e.g. CXCL12) and stimulation of bone marrow angiogenesis (e.g. CXCL8). Thirdly, at a functional level the two systems interact because the chemokine network plays a role in similar physiological processes as the MMPs, including AML cell proliferation and migration and local regulation of angiogenesis. Both the chemokine system and MMPs are currently being evaluated as targets in anti-angiogenesis/cancer therapy and may also have potential therapeutic implications in AML. This review introduces the different members of the MMP family and describes their interactions with the chemokine network and the possible involvement of MMPs together with chemokines in leukemogenesis and chemosensitivity in AML.

NF-κB usually has antiapotptic effects and is involved in regulation of cell proliferation and intercellular communication. This is also true for the malignant cells in acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS), including the malignant stem cells. However, both AML and MDS patients are heterogeneous with regard to the effect of pharmacological NF-κB inhibition, and the final effect will probably also depend on the pharmacological agent used for the inhibition, e.g. proteasomal inhibitiors versus specific inhibitors. Even though initial studies suggest that NF-κB inhibitors have antileukemic effects, their future clinical use will also depend on their toxicity profile.

Clonal heterogeneity detected by karyotyping is a biomarker associated with adverse prognosis in acute myeloid leukemia (AML). Constitutive activation of the phosphatidylinositol-3-kinase-Akt-mechanistic target of rapamycin (PI3K-Akt-mTOR) pathway is present in AML cells, and this pathway integrates signaling from several upstream receptors/mediators. We suggest that this pathway reflects biologically important clonal heterogeneity. We investigated constitutive PI3K-Akt-mTOR pathway activation in primary human AML cells derived from 114 patients, together with 18 pathway mediators. The cohort included patients with normal karyotype or single karyotype abnormalities and with an expected heterogeneity of molecular genetic abnormalities. Clonal heterogeneity reflected as pathway mediator heterogeneity was detected for 49 patients. Global gene expression profiles of AML cell populations with and without clonal heterogeneity differed with regard to expression of ectopic olfactory receptors (a subset of G-protein coupled receptors) and proteins involved in G-protein coupled receptor signaling. Finally, the presence of clonal heterogeneity was associated with adverse prognosis for patients receiving intensive antileukemic treatment. The clonal heterogeneity as reflected in the activation status of selected mediators in the PI3K-Akt-mTOR pathway was associated with a different gene expression profile and had an independent prognostic impact. Biological heterogeneity reflected in the intracellular signaling status should be further investigated as a prognostic biomarker in human AML. publishedVersion

7020Background: Bemcentinib (BGB324) is a first-in-class, oral selective inhibitor of the RTK AXL currently in ph II clinical development across several cancer types. AXL overexpression has been es...

Background and Objectives Red blood cell concentrates (RBCs) are the major blood component transfused. Although the haemoglobin content is variable, the transfusion dose is prescribed as units of red cell concentrates. Thus, by chance, large volume patients may receive a low haemoglobin dose and low volume patients may be transfused with haemoglobin-rich RBCs. The aim of this study was to evaluate whether the haemoglobin increment (grams per litre) in the patient can be predicted from the haemoglobin dose (in grams) transfused, with and without correction for estimated blood volume. If this is true, it may be possible to achieve the predicted transfusion outcome by selecting RBCs for each patient. Materials and Methods Haemodynamically stable patients scheduled for day treatment with transfusion of RBCs were recorded. A total of 52 transfusions episodes, 27 for women and 25 for men, were recorded. Blood volumes were estimated, haemoglobin content in the RBCs was measured before transfusion, and pre- and post-transfusion haemoglobin concentrations were obtained. Results The haemoglobin content of the RBCs prepared for transfusion showed a wide range, varying from 38·7 g/unit to 69·0 g/unit. There were statistically significant correlations between haemoglobin concentration in the RBCs and haemoglobin increment in patients. Conclusion Post-transfusion increment in circulating haemoglobin can be predicted from the haemoglobin content of transfused cells, but knowledge of the patient’s blood volume improves the accuracy of prediction. It may be feasible to select the high haemoglobin content RBC for patients with largest blood volume and vice versa.

The Tie-2 receptor can bind its agonistic ligand Angiopoietin-1 (Ang-1) and the potential antagonist Ang-2. Tie-2 can be expressed both by primary human acute myeloid leukaemia (AML) cells and endothelial cells, and Tie-2-blocking antibodies are now being evaluated in clinical trials for cancer treatment.We investigated the effects of Tie-2-blocking antibodies, exogenous Ang-2 and pharmacological agents on AML cell proliferation and the release of angioregulatory mediators.Tie-2-blocking antibodies had a growth inhibitory effect on human AML cells co-cultured with microvascular endothelial cells, but this inhibition was not observed when leukaemic cells were co-cultured with fibroblasts or osteoblasts. AML cell viability in co-cultures was not altered by anti-Tie-2. Furthermore, anti-Tie-2 decreased hepatocyte growth factor (HGF) levels and increased CXCL8 levels in co-cultures, whereas the levels of endocan (a proteoglycan released by endothelial cells) were not altered. The only significant effects of exogenous Ang-2 were decreased levels of HGF and endocan. Constitutive AML cell release of agonistic Ang-1 was decreased by the proteasomal inhibitor bortezomib and the specific IkappaB-kinase/NFkappaB inhibitor BMS-345541.We conclude that various strategies for inhibition of Tie-2-mediated signalling should be considered in AML therapy, possibly in combination with other antiangiogenic strategies.

Allogeneic stem cell transplantation is used in the treatment of younger patients with severe hematological diseases, especially hematological malignancies, and acute graft versus host disease (GVHD) is then an important immune-mediated posttransplant complication. Several risk factors for acute GVHD have been identified, including pretransplant factors that possibly influence the posttranspant course through their effects on host immunocompetent cells. Metabolic regulation is important for immunoregulation, and we therefore investigated whether the pretransplant metabolic status of allotransplant recipients was associated with later acute GVHD. In our population-based study we investigated the systemic (serum) metabolic profile for 86 consecutive allotransplant recipients. The samples were collected before start of the pretransplant conditioning therapy. Patients who developed later acute GVHD especially showed altered pretransplant amino acid metabolism, including (1) altered metabolism of immunoregulatory branched chain amino acids (leucine, isoleucine and valine); and (2) altered levels of potentially proinflammatory tyrosine metabolites (p-cresol sulphate, 3-phenylpropionate) formed by the gastrointestinal microbial flora. However, isobutyrylcarnitine and propyonylcarnitine levels were also altered; the carnitines are important for the transport of fatty acids and may also be important for the release of immunoregulatory cytokines in allotransplant recipients. These metabolic alterations were associated with an ongoing pretransplant acute phase reaction or early hematopoietic/immune reconstitution. Thus, allotransplant recipients developing acute GVHD showed altered preconditioning/pretransplant levels of several immunoregulatory metabolites. Our hypothesis is that these metabolites alter or activate recipient immunocompetent cells and thereby enhance or initiate anti-recipient immune reactivity. Electronic supplementary material The online version of this article (doi:10.1007/s11306-015-0880-x) contains supplementary material, which is available to authorized users.

A range of cytokines are altered during sepsis. Multiplex analysis of inflammatory mediators makes it easier to study a larger range of mediators, and combined with newer bioinformatical tools (unsupervised hierarchical clustering), this has the potential to bring new knowledge of the inflammatory process during sepsis.

Background Cytarabine is used in the treatment of acute myeloid leukemia (AML). Low-dose cytarabine can be combined with valproic acid and all-trans retinoic acid (ATRA) as AML-stabilizing treatment. We have investigated the possible risk of immunotoxicity by this combination. We examined the effects of cytarabine combined with valproic acid and ATRA on in vitro activated human T cells, and we tested cytarabine at concentrations reached during in vivo treatment with high doses, conventional doses and low doses. Methods T cells derived from blood donors were activated in vitro in cell culture medium alone or supplemented with ATRA (1 μM), valproic acid (500 or 1000 μM) or cytarabine (0.01-44 μM). Cell characteristics were assessed by flow cytometry. Supernatants were analyzed for cytokines by ELISA or Luminex. Effects on primary human AML cell viability and proliferation of low-dose cytarabine (0.01-0.5 μM) were also assessed. Statistical tests include ANOVA and Cluster analyses. Results Only cytarabine 44 μM had both antiproliferative and proapoptotic effects. Additionally, this concentration increased the CD4:CD8 T cell ratio, prolonged the expression of the CD69 activation marker, inhibited CD95L and heat shock protein (HSP) 90 release, and decreased the release of several cytokines. In contrast, the lowest concentrations (0.35 and 0.01 μM) did not have or showed minor antiproliferative or cytotoxic effects, did not alter activation marker expression (CD38, CD69) or the release of CD95L and HSP90, but inhibited the release of certain T cell cytokines. Even when these lower cytarabine concentrations were combined with ATRA and/or valproic acid there was still no or minor effects on T cell viability. However, these combinations had strong antiproliferative effects, the expression of both CD38 and CD69 was altered and there was a stronger inhibition of the release of FasL, HSP90 as well as several cytokines. Cytarabine (0.01-0.05 μM) showed a dose-dependent antiproliferative effect on AML cells, and in contrast to the T cells this effect reached statistical significance even at 0.01 μM. Conclusions Even low levels of cytarabine, and especially when combined with ATRA and valproic acid, can decrease T cell viability, alter activation-induced membrane-molecule expression and decrease the cytokine release. Electronic supplementary material The online version of this article (doi:10.1186/s40360-015-0012-2) contains supplementary material, which is available to authorized users.

Even though malnutrition is associated with an adverse prognosis after allogeneic stem cell transplantation, few studies have addressed the question what is the optimal nutritional support for these patients. There is a general agreement that the body weight, body mass index and nutritional intake can be used to guide the post-transplant nutritional support; enteral nutrition may then be tried but most patients will require parenteral nutrition. There is no scientific basis for further standardization of post-transplant nutritional support. The nutritional status with regard to amino acid as well as fatty acid metabolism and vitamin levels are important for immunoregulation. Several amino acids and their metabolites function as signaling molecules through their binding to specific receptors, and they are thereby become important both in dendritic cell differentiation and T cell activation and the metabolic switch that often occurs during the activation of immunocompetent cells. We review previous studies of nutritional support in allotransplant recipients and discuss possible molecular mechanisms involved in metabolic immunoregulation and the development of post-transplant immune-mediated complications.

Human acute myeloid leukemia is a heterogeneous disease and the effect of therapeutic targeting of specific molecular mechanisms will probably vary between patient subsets. Cell cycle regulators are among the emerging targets (e.g., aurora and polo-like kinases, cyclin-dependent kinases). Inhibition of communication between acute myeloid leukemia and stromal cells is also considered; among the most promising of these strategies are inhibition of hedgehog-initiated, CXCR4-CXCL12 and Axl-Gas6 signaling. Finally, targeting of energy and protein metabolism is considered, the most promising strategy being inhibition of isocitrate dehydrogenase in patients with IDH mutations. Thus, several strategies are now considered, and a major common challenge for all of them is to clarify how they should be combined with each other or with conventional chemotherapy, and whether their use should be limited to certain subsets of patients.

International audience; The CXXC5 gene encodes a transcriptional activator with a zinc-finger domain, and high expression in human acute myeloid leukemia (AML) cells is associated with adverse prognosis. We now characterized the biological context of CXXC5 expression in primary human AML cells. The global gene expression profile of AML cells derived from 48 consecutive patients was analyzed; cells with high and low CXXC5 expression then showed major differences with regard to extracellular communication and intracellular signaling. We observed significant differences in the phosphorylation status of several intracellular signaling mediators (CREB, PDK1, SRC, STAT1, p38, STAT3, rpS6) that are important for PI3K-Akt-mTOR signaling and/or transcriptional regulation. High CXXC5 expression was also associated with high mRNA expression of several stem cell-associated transcriptional regulators, the strongest associations being with WT1, GATA2, RUNX1, LYL1, DNMT3, SPI1, and MYB. Finally, CXXC5 knockdown in human AML cell lines caused significantly increased expression of the potential tumor suppressor gene TSC22 and genes encoding the growth factor receptor KIT, the cytokine Angiopoietin 1 and the selenium-containing glycoprotein Selenoprotein P. Thus, high CXXC5 expression seems to affect several steps in human leukemogenesis, including intracellular events as well as extracellular communication. INTRODUCTION CXXC5 is a retinoid-responsive gene localized to the 5q31.3 chromosomal region [1] and encoding a retinoid-inducible nuclear factor (RINF) [2] that is a protein containing a CXXC-type zinc-finger domain and acting as a transcription regulator [3]. Expression studies as well as gene silencing experiments suggest that CXXC5 is important in normal myelopoiesis [2] and for differentiation of endothelial cells [3]. Furthermore, we recently described that CXXC5 is expressed in primary acute myeloid leukemia (AML) cells; this expression shows a wide variation between patients and high levels are associated with an adverse prognosis and resistance to chemotherapy-induced apoptosis [4]. Another study recently confirmed our observations and CXXC5 expression was then of independent prognostic significance in multivariate analyses after adjustment

BACKGROUND Infection after arthroscopy is a rare, but feared complication. Aggressive infection with atypical microbiological agents severely complicates the clinical evaluation. CASE PRESENTATION A man in his thirties, previously healthy man had undergone elective knee arthroscopy with synovectomy. Eight days after surgery he was admitted to hospital with swelling and pain in the operated knee. Re-arthroscopy revealed bleeding in the affected joint, but no obvious signs of infection. The patient developed severe pain, fever and elevated infection parameters. Staphylococcus lugdunensis was detected in the joint fluid taken during re-arthroscopy. He was treated with a combination of antibiotics, including cloxacillin, gentamicin and linezolid, and made a full recovery after treatment. INTERPRETATION S lugdunensis is a highly virulent, coagulase-negative staphylococcus, capable of causing significant infections. It should never be dismissed as a contaminant without careful review.

Cell division cycle 25 (CDC25) protein phosphatases regulate cell cycle progression through the activation of cyclin-dependent kinases (CDKs), but they are also involved in chromatin modulation and transcriptional regulation. CDC25 inhibition is regarded as a possible therapeutic strategy for the treatment of human malignancies, including acute myeloid leukemia (AML). We investigated the in vitro effects of CDC25 inhibitors on primary human AML cells derived from 79 unselected patients in suspension cultures. Both the previously well-characterized CDC25 inhibitor NSC95397, as well as five other inhibitors (BN82002 and the novel small molecular compounds ALX1, ALX2, ALX3, and ALX4), only exhibited antiproliferative effects for a subset of patients when tested alone. These antiproliferative effects showed associations with differences in genetic abnormalities and/or AML cell differentiation. However, the responders to CDC25 inhibition could be identified by analysis of global gene expression profiles. The differentially expressed genes were associated with the cytoskeleton, microtubules, and cell signaling. The constitutive release of 28 soluble mediators showed a wide variation among patients and this variation was maintained in the presence of CDC25 inhibition. Finally, NSC95397 had no or only minimal effects on AML cell viability. In conclusion, CDC25 inhibition has antiproliferative effects on primary human AML cells for a subset of patients, and these patients can be identified by gene expression profiling. publishedVersion

The surface molecule signature of primary human acute myeloid leukemia (AML) cells is highly associated with NPM1 mutation status

Bone marrow stromal cells support both normal and malignant hematopoiesis. Τhis support is mediated through the local cytokine network and by direct cell‑cell interactions mediated via adhesion molecules and the formation of gap junctions by connexins. Previous studies on connexins in human acute myeloid leukemia (AML) have mainly focused on the investigation of leukemia cell lines. In the present study, we therefore investigated the expression of various connexins at the protein (i.e., cell surface expression) and mRNA level in primary human AML cells. The cell surface expression of the connexins, Cx26, Cx32, Cx37, Cx43 and Cx45, varied considerably between patients, and detectable levels were observed only for subsets of patients. On the whole, Cx43 and Cx45 showed the highest cell surface expression. Connexin expression was dependent on AML cell differentiation, but showed no association with cytogenetic abnormalities or mutations of the fms-related tyrosine kinase 3 (FLT3) or nucleophosmin (NPM)‑1 genes. By contrast, only Cx45 showed a significant variation between patients at the mRNA level. A high Cx45 expression was associated with the altered regulation of the mitogen‑activated protein kinase (MAPK) pathway and the release of pro-inflammatory cytokines [interleukin (IL)‑17, tumor necrosis factor (TNF), interferon‑γ], whereas a low Cx45 expression was associated with the altered regulation of protein functions (i.e., ligase activity, protein folding and catabolism). There was no significant correlation observed between the connexin mRNA and protein levels. Thus, differences in connexin expression can be used to subclassify AML patients. Differences in connexin cell surface expression profiles are not reflected at the mRNA level and have to be directly examined, whereas variations in Cx45 mRNA expression are associated with differences in cell signaling and the regulation of protein functions.

The initial evaluation of patients with suspected deep vein thrombosis includes the use of biomarkers reflecting activation of the coagulation system. However, the thromboembolic process and neighboring inflammatory responses also affect endothelial cells, and endothelial cell markers may therefore be altered by the disease. In the present population-based single-center study, we investigated the plasma levels of the endothelium-specific biomarkers soluble E-selectin and endocan in a consecutive and unselected group of 120 patients admitted to hospital for suspected deep vein thrombosis. Blood samples were collected when patients arrived at the hospital. DVT patients showed evidence for an acute phase reaction with increased serum C-reactive protein levels, but this was similar to many other patients admitted with suspected but not verified thrombosis. Plasma endocan and E-selectin levels did not differ between patients with thrombosis, healthy controls and the patients without verified thrombosis (i.e. patients with other causes of their symptoms, including various inflammatory and non-inflammatory conditions). However, the combined use of endothelial biomarkers, C-reactive protein and D-dimer could be used to identify patient subsets with different frequencies of venous thrombosis. Thus, analysis of plasma biomarker profiles including endothelial cell markers may be helpful in the initial evaluation of patients with deep vein thrombosis.

The malignant cell population of acute myeloid leukemia (AML) includes a small population of stem/progenitor cells with long-term in vitro proliferation. We wanted to compare long-term AML cell proliferation for unselected patients, investigate the influence of endothelial cells on AML cell proliferation and identify biological characteristics associated with clonogenic capacity.Cells were cultured in medium supplemented with recombinant growth factors FMS-like tyrosine kinase-3 ligand, stem cell factor, IL-3, G-CSF and thrombopoietin. The colony-forming unit assay was used to estimate the number of progenitors in AML cell populations after 35 days of culture, and microarray was used to study global gene expression profiles between AML patients.Long-term cell proliferation was observed in 7 of 31 patients, whereas 3 additional patients showed long-term proliferation after endothelial cell coculture. Patient-specific differences in constitutive cytokine release were maintained during cell culture. Patients with long-term proliferation showed altered expression in six cell cycle-related genes (HMMR, BUB1, NUSAP1, AURKB, CCNF, DLGAP5), two genes involved in DNA replication (TOP2A, RFC3) and one gene with unknown function (LHFPL2).We identified a subset of AML patients characterized by long-term in vitro cell proliferation and altered expression of cell cycle regulators that may be potential candidates for treatment of AML.

Background Cytokines, soluble adhesion molecules and metalloproteinases can be detected in human serum or plasma samples. Such systemic levels are widely used as biomarkers in epidemiological and clinical studies. Methods We prepared serum samples and three types of plasma samples (EDTA, heparin, citric acid) from 20 healthy individuals. The levels of 31 cytokines, four soluble adhesion molecules and eight matrix metalloproteinases were analyzed by Luminex technology. Results Most mediators showed detectable levels in both plasma and serum. Several mediators that can be released by platelets showed increased serum levels, especially CCL5 and CD40L, but for the other mediators the serum levels did not correlate with peripheral blood platelet counts and for these last mediators serum and plasma levels often showed strong correlations. The use of bivalirudin for anticoagulation significantly increased and citric acid combined with platelet inhibitors (ticagrelor, acetylsalicylic acid plus prostaglandin E 2 ) did not alter plasma levels of platelet-store mediators compared with citric acid alone. The impact of sample preparation differed between mediators; for many mediators strong correlations were seen between serum and plasma levels even when absolute levels differed. Soluble adhesion molecule levels showed only minor differences between samples. Unsupervised hierarchical clustering suggested that the effect of sampling/preparation was strongest for serum and heparin plasma samples. Conclusion Careful standardization of sample preparation is usually necessary when analyzing systemic mediator levels, and differences caused by sample preparation should be considered as a possible explanation if studies show conflicting results.

Heat shock proteins (HSPs) are molecular chaperones that stabilize folding and conformation of mature proteins. HSPs are, therefore, considered as possible therapeutic targets in the treatment of human cancers, including acute myeloid leukemia (AML). This strategy offers the possibility of targeting several oncogenic proteins or several intracellular signaling pathways through the use of a single therapeutic agent.Several specific HSP70 inhibitors have now been developed. We describe: i) the molecular structure of HSP70 and the molecular interactions especially with its co-chaperones; ii) the immunoregulatory functions of HSP70; iii) the expression and function of HSP70 in human AML cells; iv) the molecular and pharmacological characteristics of potential HSP70 inhibitors; and v) the clinical and experimental studies of HSP70 inhibition in human AML. Our review is based on careful selection of relevant publications identified in the PubMed database.HSP70 inhibitors have antileukemic activity in human AML. However, additional experimental studies using in vitro models as well as animal models are required, including detailed toxicology studies, as a scientific basis for the optimal design of future clinical studies.

Acute promyelocytic leukaemia (APL) is a subtype of acute myeloid leukaemia (AML) with unique biological and clinical features and unique therapeutic requirements. The article provides a brief description of the development, pathophysiology, diagnosis and treatment of APL.The article is based on the authors' own experience and reviews of key articles and national and international guidelines.The disease is caused by a single genetic event, namely the translocation t(15;17), which gives rise to the oncoprotein PML-RARA. Clinical and morphological characteristics arouse suspicion of the disease, and the diagnosis is verified by detecting the translocation. At the time of diagnosis most patients have severe coagulopathy and the predominant clinical manifestation is bleeding. Early mortality is due to severe haemorrhage, usually intracranial. Early treatment start with all-trans retinoic acid (ATRA) on suspicion of APL is essential to reduce this early mortality. ATRA is also an important part of continued treatment, in combination with anthracycline-based chemotherapy and possibly arsenic. After this treatment, the prognosis for disease-free long-term survival is90%. There are also safe and effective treatment options for elderly patients with complex comorbidities.With APL it is particularly important to start disease-targeting therapy in the form of ATRA quickly because of the high risk of serious haemorrhages and high early mortality. If serious haemorrhages are avoided, the prognosis is very good.

Endothelial cells are involved in the pathogenesis of acute graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. These cells express several molecules that can be detected as biologically active soluble forms; serum levels of these molecules may thereby reflect the functional status of endothelial cells. Furthermore, acute GVHD is an inflammatory reaction and endothelial cells function as local regulators of inflammation. We therefore investigated whether differences in preconditioning/pretransplant serum levels of endothelium-expressed molecules (i.e., endocan, vascular cell adhesion molecule 1 (VCAM-1), and E-selectin) were associated with a risk of posttransplant GVHD. Our study should be regarded as a population-based study of consecutive and thereby unselected patients (n=56). Analysis of this pretreatment endothelium biomarker profile by unsupervised hierarchical clustering identified a subset of patients with increased early nonrelapse mortality. Furthermore, low endocan levels were significantly associated with acute GVHD in the liver and gastrointestinal tract, whereas high VCAM-1 levels were associated with acute GVHD in the skin only. Our study suggests that the preconditioning/pretransplant status of endothelial cells (possibly through altered trafficking of immunocompetent cells) is important for the risk and the organ involvement of later acute GVHD.

Background: The small-molecule MDM2 antagonist nutlin-3 has proved to be an effective p53 activating therapeutic compound in several preclinical cancer models, including acute myeloid leukemia (AML). We and others have previously reported a vigorous acetylation of the p53 protein by nutlin-treatment. In this study we aimed to investigate the functional role of this p53 acetylation in nutlin-sensitivity, and further to explore if nutlin-induced protein acetylation in general could indicate novel targets for the enhancement of nutlin-based therapy. Results: Nutlin-3 was found to enhance the acetylation of p53 in the human AML cell line MOLM-13 (wild type TP53) and in TP53 null cells transfected with wild type p53 cDNA. Stable isotope labeling with amino acids in cell culture (SILAC) in combination with immunoprecipitation using an anti-acetyl-lysine antibody and mass spectrometry analysis identified increased levels of acetylated Histone H2B, Hsp27 and Hsp90 in MOLM-13 cells after nutlin-treatment, accompanied by downregulation of total levels of Hsp27 and Hsp90. Intrac ellular levels of heat shock proteins Hsp27, Hsp40, Hsp60, Hsp70 and Hsp90α were correlated to nutlin-sensitivity for primary AML cells (n = 40), and AML patient samples with low sensitivity to nutlin-3 tended to express higher levels of heat shock proteins than more responsive samples. Combination therapy of nutlin-3 and Hsp90 inhibitor geldanamycin demonstrated synergistic induction of apoptosis in AML cell lines and primary AML cells. Finally, TP53 null cells transfected with a p53 acetylation defective mutant demonstrated decreased heat shock protein acetylation and sensitivity to nutlin-3 compared to wild type p53 expressing cells. Conclusions: Altogether, our results demonstrate that nutlin-3 induces acetylation of p53, histones and heat shock proteins, and indicate that p53 acetylation status and the levels of heat shock proteins may participate in modulation of nutlin-3 sensitivity in AML. publishedVersion

Extracorporeal photopheresis (ECP) is an immunomodulatory alternative for treatment of graft versus host disease (GVHD). The blood is then separated into its various components through apheresis; buffy coat cells are thereafter treated with 8-methoxypsoralen before exposure to ultraviolet light and finally reinfused into the patient. There is a general agreement that this treatment has an anti-GVHD effect, but the mechanisms of action behind this effect are only partly understood. However, altered maturation of dendritic cells (DC) and thereby indirect modulation of T-cell reactivity seems to be one important mechanism together with DC-presentation of antigens derived from apoptotic donor T cells and induction of regulatory T cells. The treatment has been best studied in patients with chronic GVHD (both pediatric and adult patients), but most studies are not randomized and it is difficult to know whether the treatment is more effective than the alternatives. The clinical studies of ECP in adults with acute GVHD are few and not randomized; it is not possible to judge whether this treatment should be a preferred second- or third-line treatment. There is no evidence for increased risk of leukemia relapse or suppression of specific graft versus leukemia reactivity by this treatment, so specific antileukemic immunotherapy may still be possible. Thus, even though the treatment seems effective in patients with GVHD, further clinical (especially randomized) as well as biological studies with careful standardization of the treatment are needed before it is possible to conclude how ECP should be used in acute and chronic GVHD.

Acute myeloid leukaemia (AML) is a heterogeneous malignancy. Intracellular signalling through the phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR) pathway is important for regulation of cellular growth and metabolism, and inhibitors of this pathway is considered for AML treatment. Primary human AML cells, derived from 96 consecutive adult patients, were examined. The effects of two mTOR inhibitors (rapamycin, temsirolimus) and two PI3K inhibitors (GDC-0941, 3-methyladenine) were studied, and we investigated cytokine-dependent proliferation, regulation of apoptosis and global gene expression profiles. Only a subset of patients demonstrated strong antiproliferative effects of PI3K-mTOR inhibitors. Unsupervised hierarchical clustering analysis identified two main clusters of patients; one subset showing weak or absent antiproliferative effects (59%) and another group showing a strong growth inhibition for all drugs and concentrations examined (41%). Global gene expression analyses showed that patients with AML cell resistance against PI3K-mTOR inhibitors showed increased mRNA expression of the CDC25B gene that encodes the cell cycle regulator Cell Division Cycle 25B. The antileukaemic effect of PI3K-Akt-mTOR inhibition varies between patients, and resistance to these inhibitors is associated with the expression of the cell cycle regulator CDC25B, which is known to crosstalk with the PI3K-Akt-mTOR pathway and mediate rapamycin resistance in experimental models.

Acute myeloid leukemia (AML) is characterized by bone marrow accumulation of immature leukemic blast cells. Conventional AML treatment includes induction chemotherapy to achieve disease control, followed by consolidation therapy with conventional chemotherapy or allogeneic/autologous stem cell transplantation (allo/auto-SCT) to eradicate residual disease. Even younger patients receiving the most intensive treatment have a median, long-term, AML-free survival of only 45-50%, highlighting the need for new treatment strategies. The important role of the bone marrow cytokine network during disease development and treatment is suggested by several observations, including: (i) the increased microvascular density (MVD) in leukemic bone marrow, (ii) experimental evidence of cytokine-mediated crosstalk between leukemic and microvascular endothelial cells, (iii) the prognostic impact of angioregulatory cytokine levels both in patients receiving conventional chemotherapy and allo-SCT, and (iv) the experimental evidence for an antileukemic effect of cytokine inhibition in human AML. Several cytokines are constitutively released by human AML cells, including interleukins, chemokines, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and angiopoietins. However, the cytokine system constitutes a functional, interacting network, and recent evidence suggests that analysis of serum cytokine profiles rather than the analysis of single cytokines should be used for prognostic evaluation of AML patients. We will discuss the role of angioregulatory cytokines in leukemogenesis, including their direct effects on the leukemic cells, as well as their indirect contribution to leukemogenesis through angioregulation and crosstalk between leukemic and neighboring stromal cells. We shall also discuss the possibility of targeting angioregulatory cytokines as a part of the treatment strategy in leukemia.

Axl is a receptor tyrosine kinase that has been shown to have a strong oncogenic potential in many cancer types. Overexpression and activation of Axl is found in many cancers, and is linked to increased proliferation, migration/invasion and resistance to apoptosis. Axl overexpression has been shown to be a poor prognostic marker, and recently overexpression of Axl has also been linked to the acquired resistance to chemotherapy and other anticancer therapies in many malignancies, including AML. BGB324 (BerGenBio AS) is a first-in-class highly specific small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in a clinical safety trial in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in a phase 1b clinical trial in patients with refractory/relapsed AML and MDS (BGBC003, ClinicalTrials.gov Identifier:NCT02488408; Loges S et al. J Clin Oncol 34, 2016 suppl; abstr 2561). 20 AML and 4 MDS patients have been treated at the following dose levels (loading dose/continuation dose): 400/100mg, 600/200mg and 900/300mg. Objective responses were observed in 2/4 MDS patients and 2/20 AML patients including one CR (AML). Enrollment continues to define MTD. The effect of BGB324 on intracellular signaling and the immune profile of leukemic blasts in patients treated in the clinical study was investigated using phospho-flow cytometry. Blasts were identified using surface markers (CD45low, CD66b-, CD38-, and CD117+ and/or CD34+), and the following direct and indirect downstream targets of Axl were explored: phosphorylated (p)-Akt(S473 and T308), pErk(T202/Y204), pp38(T180/Y182), pPLCγ1(Y783), pNFκB(S529), pCREB(S113) and pSTAT1(Y701), 3(Y705), 5(Y694)and 6(Y641). Preliminary analyses of blood samples from six patients show very rapid responses in signaling pathways downstream of Axl (including Akt, Erk, NFκB and PLCγ1) within hours or days of ingestion of the first dose, although the response patterns varies from patient to patient (Figure 1A). Two distinct blast populations were identified: one CD117+/CD34- and one CD117+/CD34+. In most patients the CD117+/CD34- population displayed the most extensive signaling changes during treatment, and this population also decreased during treatment with BGB324. In contrast, the CD117+/CD34+ population expanded during the course of the treatment (Figure 1B). White cell differential counts of peripheral blood from two patients treated with BGB324 for a prolonged period of time (15 weeks or more) showed a decrease in peripheral blast count, and a corresponding increase in granulocyte and monocyte counts, suggesting that Axl inhibition may push the blasts towards differentiation. The clinical trial is ongoing, and the signaling profile of leukemic blasts in blood and bone marrow of treated patients will be further examined by conventional phosphoflow cytometry and mass cytometry searching for signaling profiles with prognostic information. In conclusion, BGB324 has unique pharmacodynamic properties and molecular responses to exposure can be observed in peripheral blood leukemic blasts by phospho-flow cytometry within hours of ingestion of the first treatment dose. Further studies may establish whether single cell signal profiling can discriminate responders from non-responders and provide information about dose-response in a clinically meaningful way. Disclosures Cortes: Astellas: Research Funding; Arog: Research Funding; Teva: Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Ariad: Consultancy, Research Funding; Ambit: Research Funding. Heuser:Novartis: Consultancy, Research Funding; Tetralogic: Research Funding; BerGenBio: Research Funding; Karyopharm Therapeutics Inc: Research Funding; Bayer Pharma AG: Research Funding; Celgene: Honoraria; Pfizer: Research Funding. Lorens:BerGenBio AS: Employment, Equity Ownership, Research Funding. Gausdal:BerGenBio AS: Employment. Micklem:BerGenBio AS: Employment, Equity Ownership. Gjertsen:BerGenBio AS: Consultancy, Research Funding.

In patients who have large bleeds, there is a tendency to transfuse more plasma and platelets than recommended in earlier guidelines, and accordingly many hospitals now provide "transfusion packages" with an intended red cell:platelet:plasma ratio of 1:1:1. The purpose of this study was to investigate in vitro functions of transfusion packs compared with fresh whole blood."Reconstituted whole blood" was prepared with the same ratio of red cells, platelets and plasma as used in local transfusion packages. The aggregation and thrombin-antithrombin complex formation responses to collagen stimulation of this reconstituted whole blood were compared with those of fresh whole blood. The storage time of red cells and platelets was varied in a systematic manner, giving nine different compositions of reconstituted whole blood that simulated transfusion packs.The responses varied significantly between whole blood and reconstituted whole blood -and between the reconstituted whole blood of different compositions. A significant decrease (p0.005) in collagen-induced platelet count reduction was seen with increasing platelet and red blood cell age. Thrombin-antithrombin complex formation peaked in studies with platelets stored for 5 days. The red cells stored for the longest time induced the greatest thrombin-antithrombin complex formation. Fresh whole blood gives more consistent responses, and the aggregation response to collagen is stronger than in reconstituted whole blood.Our results indicate that in vitro responses of reconstituted whole blood vary substantially according to how long the red cells and platelets are stored for. As the responses obtained by testing whole blood are more consistent and usually stronger, the alternative use fresh whole blood in special conditions should not be excluded without further consideration.

Effects of the mTOR inhibitor rapamycin were characterized on in vitro cultured primary human acute myeloid leukemia (AML) cells and five AML cell lines. Constitutive mTOR activation seemed to be a general characteristic of primary AML cells. Increased cellular stress induced by serum deprivation increased both mTOR signaling, lysosomal acidity, and in vitro apoptosis, where lysosomal acidity/apoptosis were independent of increased mTOR signaling. Rapamycin had antiproliferative and proapoptotic effects only for a subset of patients. Proapoptotic effect was detected for AML cell lines only in the presence of serum. Combination of rapamycin with valproic acid, all-trans retinoic acid (ATRA), and NF-κB inhibitors showed no interference with constitutive mTOR activation and mTOR inhibitory effect of rapamycin and no additional proapoptotic effect compared to rapamycin alone. In contrast, dual inhibition of the PI3K-Akt-mTOR pathway by rapamycin plus a PI3K inhibitor induced new functional effects that did not simply reflect a summary of single drug effects. To conclude, (i) pharmacological characterization of PI3K-Akt-mTOR inhibitors requires carefully standardized experimental models, (ii) rapamycin effects differ between patients, and (iii) combined targeting of different steps in this pathway should be further investigated whereas combination of rapamycin with valproic acid, ATRA, or NF-κB inhibitors seems less promising.

Increased bone marrow angiogenesis is seen in several hematological malignancies, including acute myeloid leukemia (AML). We used a co-culture assay of endothelial and vascular smooth muscle cells (vSMC) to investigate the effects of AML-conditioned medium on capillary networks. We investigated primary AML cells derived from 44 unselected patients and observed that for a large subset of patients, the constitutive cytokine release by the leukemic cells stimulated endothelial cell organization into capillary-like networks, while there were only minor or no effects for other patients. We analyzed the constitutive AML cell release of 31 cytokines for all the patients and performed a hierarchical cluster analysis of the cytokine profile which identified two major patient subsets that differed in their ability to enhance capillary-like networks; increased capillary-like networks was then associated with high constitutive release of several cytokines and especially high levels of several pro-angiogenic chemokines. Significantly increased network formation was not seen for any of the 11 acute lymphoblastic leukemia patients investigated. The cytokine response by activated normal T cells inhibited endothelial network formation in our in vitro model of angiogenesis and activated normal monocytes had only a minor influence on tube formation. Our study shows that AML-derived cytokines can induce the organization of endothelial cells into vessel-like structures.

Infection after arthroscopy is a rare, but feared complication. Aggressive infection with atypical microbiological agents severely complicates the clinical evaluation.A man in his thirties, previously healthy man had undergone elective knee arthroscopy with synovectomy. Eight days after surgery he was admitted to hospital with swelling and pain in the operated knee. Re-arthroscopy revealed bleeding in the affected joint, but no obvious signs of infection. The patient developed severe pain, fever and elevated infection parameters. Staphylococcus lugdunensis was detected in the joint fluid taken during re-arthroscopy. He was treated with a combination of antibiotics, including cloxacillin, gentamicin and linezolid, and made a full recovery after treatment.S lugdunensis is a highly virulent, coagulase-negative staphylococcus, capable of causing significant infections. It should never be dismissed as a contaminant without careful review.

Five human polo-like kinases (PLKs) have been identified, and PLK1 - 4 seem to interact with Aurora kinases and act as cell cycle regulators in both normal and malignant human cells.The present review describes i) experimental evidence for a role for PLKs and Aurora kinases in human leukemogenesis and ii) the results from clinical studies of PLK and Aurora kinase inhibitors in the treatment of human acute myeloid leukemia (AML). The review was based on searches in the PubMed and the ClinicalTrials.gov databases. These inhibitors have antiproliferative and proapoptotic effects in AML cells. Hematological and gastrointestinal toxicities are frequently dose limiting, and this may limit the use of these agents in combination with conventional AML therapy. Aurora kinase inhibitors seem to be most effective for patients with high expression of the target kinases, and the same may be true for PLK inhibitors.PLK inhibition is a promising strategy for the treatment of AML. Future clinical studies have to clarify i) whether this strategy is most effective for certain subsets of patients; ii) whether multikinase inhibitors targeting several cell cycle regulators should be preferred; and iii) how this therapeutic strategy eventually should be combined with conventional antileukemic chemotherapy.

Voluntary donation is a key issue in transfusion medicine. To ensure the safety of blood transfusions, careful donor selection is important. Although new approaches to blood safety have dramatically reduced the risks for infectious contamination of blood components, the quality and the availability of blood components depend on the willingness to donate and the reliability of the information given by the donors about their own health, including risk behavior. As donors who are deferred by the blood bank will be less motivated to return for donation, it is important to reduce the number of deferrals. The aims of the present study were to investigate the reasons for deferral of registered donors coming to the blood bank for donation, in order to identify areas of importance for donor education—as these deferrals potentially could be avoided by better donor comprehension. Deferral related to testing of donors is not included in this study as these deferrals are dependent on laboratory results and cannot be indentified by questionnaire or interview. Data were collected from all blood donors in a period for 18 months who came for blood donation at a large university hospital in Norway. 1 163 of the 29 787 regular donors, who showed up for donation, were deferred (3.9%). The main reasons were intercurrent illness (n=182) (15.6%), skin ulcers (n=170) (14.6%), and risk behaviour (n=127) (10.9%). In a community, intercurrent illnesses, skin ulcers, and potential risk behavior are the most frequent reasons for deferral of regular donors. Strategized effort on donor education is needed, as “failure to donate” reduces donor motivation.

The t(8;21) abnormality occurs in a minority of acute myeloid leukemia (AML) patients. The translocation results in an in-frame fusion of two genes, resulting in a fusion protein of one N-terminal domain from the AML1 gene and four C-terminal domains from the ETO gene. This protein has multiple effects on the regulation of the proliferation, the differentiation, and the viability of leukemic cells. The translocation can be detected as the only genetic abnormality or as part of more complex abnormalities. If t(8;21) is detected in a patient with bone marrow pathology, the diagnosis AML can be made based on this abnormality alone. t(8;21) is usually associated with a good prognosis. Whether the detection of the fusion gene can be used for evaluation of minimal residual disease and risk of leukemia relapse remains to be clarified. To conclude, detection of t(8;21) is essential for optimal handling of these patients as it has both diagnostic, prognostic, and therapeutic implications.

The human Notch system consists of 5 ligands and 4 membrane receptors with promiscuous ligand binding, and Notch-initiated signalling interacts with a wide range of other intracellular pathways. The receptor signalling seems important for regulation of normal and malignant hematopoiesis, development of the cellular immune system, and regulation of immune responses. Several Notch-targeting agents are now being developed, including natural receptor ligands, agonistic and antagonistic antibodies, and inhibitors of intracellular Notch-initiated signalling. Some of these agents are in clinical trials, and several therapeutic strategies seem possible in stem cell recipients: (i) agonists may be used for stem cell expansion and possibly to enhance posttransplant lymphoid reconstitution; (ii) receptor-specific agonists or antagonists can be used for immunomodulation; (iii) Notch targeting may have direct anticancer effects. Although the effects of therapeutic targeting are difficult to predict due to promiscuous ligand binding, targeting of this system may represent an opportunity to achieve combined effects with earlier posttransplant reconstitution, immunomodulation, or direct anticancer effects.

Acute myelogenous leukemia (AML) is a bone marrow disease in which the leukemic cells show constitutive release of a wide range of CCL and CXCL chemokines and express several chemokine receptors. The AML cell release of various chemokines is often correlated and three release clusters have been identified: CCL2-4/CXCL1/8, CCL5/CXCL9-11, and CCL13/17/22/24/CXCL5. CXCL8 is the chemokine usually released at highest levels. Based on their overall constitutive release profile, patients can be classified into distinct subsets that differ in their T cell chemotaxis towards the leukemic cells. The release profile is modified by hypoxia, differentiation status, pharmacological interventions, and T cell cytokine responses. The best investigated single chemokine in AML is CXCL12 that binds to CXCR4. CXCL12/CXCR4 is important in leukemogenesis through regulation of AML cell migration, and CXCR4 expression is an adverse prognostic factor for patient survival after chemotherapy. Even though AML cells usually release high levels of several chemokines, there is no general increase of serum chemokine levels in these patients and the levels are also influenced by patient age, disease status, chemotherapy regimen, and complicating infections. However, serum CXCL8 levels seem to partly reflect the leukemic cell burden in AML. Specific chemokine inhibitors are currently being developed, although redundancy and pleiotropy of the chemokine system are obstacles in drug development.

Acute myeloid leukemia (AML) is an aggressive malignancy with only 40%-50% long-term survival even for younger patients who can receive the most aggressive therapy. For elderly patients who only receive palliative treatment, the median survival is only 2-3 months. Inhibition of the nuclear factor-kappaB (NF-kappaB) transcription factor family is one of the therapeutic strategies that are considered in AML. NF-kappaB is an important regulator of several biological processes that are involved in leukemogenesis, including proliferation, differentiation, autophagy, and apoptosis. Constitutive NF-kappaB activation has been detected in AML cells and NF-kappaB inhibition is therefore a possible therapeutic strategy in AML. Multiple pharmacological agents have shown inhibitory effects against NF-kappaB signaling pathways, including proteasome inhibitors as well as the more-specific agents that are directed against various steps of this signaling pathway. Recent studies strongly suggest that primary human AML cells (including AML stem cells) are susceptible to NF-kappaB inhibition, but this therapeutic approach should possibly be combined with other therapeutic agents to achieve a combined effect both on NF-kappaB transcriptional activity, tumor suppressor-induced signaling, and stress-induced pathways. The clinical documentation with regard to the efficiency and safety of NF-kappaB inhibition is still limited, but experimental evidence strongly suggests that NF-kappaB inhibition should be further investigated in human AML.

Acute myelogenous leukemia (AML) is a bone marrow disease in which the leukemic cells show constitutive release of a wide range of CCL and CXCL chemokines and express several chemokine receptors. The AML cell release of various chemokines is often correlated and three release clusters have been identified: CCL2-4/CXCL1/8, CCL5/CXCL9-11, and CCL13/17/22/24/CXCL5. CXCL8 is the chemokine usually released at highest levels. Based on their overall constitutive release profile, patients can be classified into distinct subsets that differ in their T cell chemotaxis towards the leukemic cells. The release profile is modified by hypoxia, differentiation status, pharmacological interventions, and T cell cytokine responses. The best investigated single chemokine in AML is CXCL12 that binds to CXCR4. CXCL12/CXCR4 is important in leukemogenesis through regulation of AML cell migration, and CXCR4 expression is an adverse prognostic factor for patient survival after chemotherapy. Even though AML cells usually release high levels of several chemokines, there is no general increase of serum chemokine levels in these patients and the levels are also influenced by patient age, disease status, chemotherapy regimen, and complicating infections. However, serum CXCL8 levels seem to partly reflect the leukemic cell burden in AML. Specific chemokine inhibitors are currently being developed, although redundancy and pleiotropy of the chemokine system are obstacles in drug development.

Objectives: Angiogenesis seems important for both leukemogenesis and chemosensitivity in acute myelogenous leukemia (AML). Angiogenesis is regulated by the balance between pro- and antiangiogenic cytokines, which also indicates an important role of matrix metalloproteases (MMPs) and their natural inhibitors, tissue inhibitors of metalloproteases (TIMPs). We investigated the constitutive release of MMPs and TIMPs for a large group of consecutive AML patients. Methods: AML cells were cultured in vitro either alone or together with microvascular endothelial cells, and levels of MMPs and TIMPs were determined in culture supernatants. Results: AML cells showed constitutive release of several MMPs and TIMPs. For all patients, detectable MMP-10 release was observed, and most patients showed detectable release of at least one additional MMP, usually MMP-9 or MMP-2. A significant correlation was found between MMP-9 and TIMP-1 release and the release of several CCL and CXCL chemokines. MMP-9 release was higher for AML cells with monocytic differentiation corresponding to the FAB-subtype M4/M5 AML; it was mainly released in its inactive form, but endogenously active MMP-9 could be detected even in the presence of the constitutively released TIMP-1/2. Endothelial cells released relatively high levels of MMP-10, and these levels were further increased by coculture with AML cells. Patients achieving complete hematological remission after only one induction cycle showed relatively low constitutive MMP-2 release. Conclusion: We conclude that primary human AML cells show constitutive release of both MMPs and TIMPs, and this release may be important for leukemogenesis and possibly also for chemosensitivity.

Heat shock proteins (HSPs) are molecular chaperones that stabilize folding and conformation of normal as well as oncogenic proteins. These chaperones thereby prevent the formation of protein aggregates. HSPs are often overexpressed in human malignancies, including AML. HSP90 is the main chaperon required for the stabilization of multiple oncogenic kinases involved in the development of acute myelogenous leukemia (AML). HSP90 client proteins are involved in the regulation of apoptosis, proliferation, autophagy and cell cycle progression; several of these proteins are in addition considered as possible therapeutic targets for the treatment of AML. HSP90 inhibition thereby offers the possibility to modulate several intracellular regulatory pathways through targeting of a single molecule. Several direct inhibitors of HSP90 have been developed, and they are classified into four groups: benzoquinon ansamycines and their derivatives, radicicol and its derivates, small synthetic inhibitors and a final group of other inhibitors. The HSP90 activity is regulated by posttranscriptional modulation; HSP90 inhibition can thereby be indirectly achieved through increased acetylation caused by histone deacetylase inhibitors. Many of these agents have entered phase I/II clinical trials, and the results from these initial studies have documented that HSP90 inhibition can mediate antileukemic effects in vivo. However, one would expect immunosuppressive side effects because HSP90 inhibitors have both direct and indirect inhibitory effects on T cell activation. Thus, future clinical studies are needed to clarify the efficiency and toxicity of HSP90 inhibitors in the treatment of human AML, including studies where HSP90 inhibitors are combined with conventional chemotherapy.