ALBERT

Beschreibung: PU.1, IKAROS, E2A, EBF, and PAX5 comprise a transcriptional network that orchestrates B-cell lineage specification, commitment, and differentiation. Here we identify interferon regulatory factor 8 (IRF8) as another component of this complex, and show that it also modulates lineage choice by hematopoietic stem cells (HSCs). IRF8 binds directly to an IRF8/Ets consensus sequence located in promoter regions of Sfpi1 and Ebf1, which encode PU.1 and EBF, respectively, and is associated with transcriptional repression of Sfpi1 and transcriptional activation of Ebf1. Bone marrows of IRF8 knockout mice (IRF8−/−) had significantly reduced numbers of pre-pro-B cells and increased numbers of myeloid cells. Although HSCs of IRF8−/− mice failed to differentiate to B220+ B-lineage cells in vitro, the defect could be rescued by transfecting HSCs with wild-type but not with a signaling-deficient IRF8 mutant. In contrast, overexpression of IRF8 in HSC-differentiated progenitor cells resulted in growth inhibition and apoptosis. We also found that IRF8 was expressed at higher levels in pre-pro-B cells than more mature B cells in wild-type mice. Together, these results indicate that IRF8 modulates lineage choice by HSCs and is part of the transcriptional network governing B-cell lineage specification, commitment, and differentiation.

Beschreibung: A distinct feature of multiple myeloma (MM) is the tight interaction between malignant plasma cells and their bone microenvironment, creating a niche suitable for MM growth. In particular, MM cells inhibit osteoblast (OB) differentiation and stimulate osteoclast (OC) function, resulting in imbalanced bone remodeling and osteolytic bone disease. Here we studied a novel cytokine, activin A, identified from a broad range of cytokines, in the development of MM bone disease. We next asked whether activin A inhibition could restore bone balance and suppress tumor growth. Activin, a member of the TNF-α superfamily, is a pleiotropic cytokine involved in bone remodeling. Here, we observed, that MM patients with multiple osteolytic lesions had a 4-fold increase in activin A expression levels in bone marrow plasma compared to MM patients with one or less osteolytic lesions and non-MM patients (average 123.6 ± 136 vs 26.4 ± 21.4 vs 30.6 ± 25.1 pg/ml respectively, p

Beschreibung: INTRODUCTION: Proteasome inhibitors (PI) and histone deacetylase inhibitors (HDACi) have demonstrated synergistic pre-clinical activity in multiple myeloma (MM). The goals of this study were to evaluate this combination regimen’s clinical activity and adverse events, including thrombocytopenia (TCP) since both drug classes may cause transient TCP. We performed an open label, single-centre, single-arm, phase I/II, dose-escalation trial of bortezomib, dexamethasone and romidepsin (depsipeptide) in relapsed or refractory MM. This is the first clinical trial to combine these 3 agents. METHODS. All patients (pts) received bortezomib (1.3mg/m2 d1, 4, 8, 11) with dexamethasone (20mg d1, 2, 4, 5, 8, 9, 11, 12). Romidepsin commenced at 8 mg/m2 IV d1, 8, and 15 every 28 days with a planned accelerated intra-patient dose escalation to 10, 12 and 14 mg/m2 (n=10). After CR + 2 cycles or a maximum of 8 cycles, pts with SD or better commenced maintenance (Mx) therapy, romidepsin at the MTD on days 1 and 8 of a 28 day cycle until PD. An additional 15 pts were treated at the MTD in a phase II expansion. Response was assessed after every 2 cycles according to IMWG criteria (with minimal Response (MR) defined as ≥25% but

Beschreibung: Endothelial cells secrete prothrombotic ultralarge von Willebrand factor (VWF) multimers, and the metalloprotease ADAMTS13 cleaves them into smaller, less dangerous multimers. This reaction is stimulated by tensile force applied to the VWF substrate, which may occur on cell surfaces or in the circulating blood. The cleavage of soluble VWF by ADAMTS13 was accelerated dramatically by a combination of platelets and fluid shear stress applied in a cone-plate viscometer. Platelet-dependent cleavage of VWF was blocked by an anti-GPIbα monoclonal antibody or by a recombinant soluble fragment of GPIbα that prevents platelet-VWF binding. Multimeric gel analysis showed that shear and platelet-dependent cleavage consumed large VWF multimers. Therefore, ADAMTS13 preferentially acts on platelet-VWF complexes under fluid shear stress. This reaction is likely to account for a majority of VWF proteolysis after secretion and to determine the steady-state size distribution of circulating VWF multimers in vivo.

Beschreibung: The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

Beschreibung: Venous thromboembolism (VTE) is increasingly diagnosed among individuals with hematologic malignancies. However, the risk of VTE among patients undergoing hematopoietic stem cell transplantation (HSCT) is unclear. We examined the incidence and risk factors for VTE and bleeding among 1514 patients undergoing in-patient HSCT. No protocolized VTE prophylaxis was used. By HSCT day 180, 75 symptomatic VTE occurred in 70 patients (4.6%; 95% confidence interval [CI], 3.6%-5.8%). Fifty-five (3.6%) were catheter-associated, 11 (0.7%) were non–catheter-associated deep venous thromboses, and 9 (0.6%) were pulmonary emboli. Thirty-four percent of VTE occurred at a platelet count less than 50 ×109/L; 13% occurred at a platelet count less than 20 ×109/L. In multivariate analysis, VTE was associated with prior VTE (odds ratio [OR], 2.9; 95% CI, 1.3-6.6) and with graft-versus-host disease (GVHD; OR, 2.4; 95% CI, 1.4-4.0). Clinically significant bleeding occurred in 230 patients (15.2%; 95% CI, 13.4%-17.1%); 55 patients (3.6%; 95% CI, 2.7%-4.7%) had fatal bleeding. Bleeding was associated with anticoagulation (OR, 3.1; 95% CI, 1.8-5.5), GVHD (OR, 2.4; 95% CI, 1.8-3.3), and veno-occlusive disease (OR, 2.2; 95% CI, 1.4-3.6). In HSCT patients, VTE is primarily catheter-related and 3-fold less common than clinically significant bleeding. These findings warrant consideration when selecting VTE prophylaxis in HSCT patients.

Beschreibung: Donor cell expression of C3 enhances the alloimmune response and is associated with the fate of transplantation. To clarify the mechanism for enhancement of the immune response, we have explored the role of C3a receptor (C3aR)–ligand interaction on murine bone marrow dendritic cells (DCs). We show that DCs either lacked receptor for C3a (a C3 cleavage product) or were treated with C3aR antagonist, elicited defective T-cell priming against alloantigen expressed on the DCs. This was associated with reduced surface expression of major histocompatibility complex (MHC) and costimulatory molecules on the DCs, and with defective priming in skin allograft rejection. In addition, DCs lacking factor B were unable to generate potent T-cell responses against donor antigen, whereas lack of C4 had no detectable effect, suggesting a role for the alternative pathway contributing to allostimulation. Furthermore, therapeutic complement regulator can down-regulate DC allostimulatory function. These findings suggest that the capacity of DCs for allostimulation depends on their ability to express, activate, and detect relevant complement components leading to C3aR signaling. This mechanism, in addition to underpinning the cell-autonomous action of donor C3 on allostimulation, has implications for a wider range of immune responses in self-restricted T-cell priming.

Beschreibung: A correlation between increase in bone markers (alkaline phosphatase (ALP) and response to bortezomib in patients with multiple myeloma (MM) has been previously described. We now report results from a prospective study examining the relationship of serum PTH variation with skeletal effects and myeloma response to bortezomib treatment. Methods: Single agent bortezomib (1.3 mg/m2 patients 1–10; 1mg/m2 patient 11–20), was administered to patients with relapsed/refractory MM on days 1, 4, 8 and 11 on a 21 day interval for a total of 3 cycles; patients were not allowed to receive concurrent bisphosphonates or any other anti-myeloma drugs during the study period. Dynamic indices of bone turnover were prospectively evaluated by high-resolution microCT. Architectural parameters such as bone volume/total volume (BVTV), trabecular number (TbN), and thickness (Tb.Th) was obtained. PTH along with bone markers (osteocalcin, calcium, magnesium, phosphorus and serum creatinine) were measured on days 1, 4, 8, 11 before and after each bortezomib dose and every 4 hours thereafter, daily for the other days of the treatment cycle. Results: Seventeen patients were enrolled in the study with a median age of 63 years, 41 % were male and 3/4 of the patients previously failed high-dose chemotherapy. Histomorphometric microCT comparative analysis was completed (baseline and post-treatment) in 7 of the 17 patients enrolled in the trial. Baseline BV/TV values ranged from 13% to 90%. After 3 cycles of bortezomib treatment a statistically significant increase in BV/TV was recorded in 6 of 7 patients (P

Beschreibung: A severely reduced ADAMTS13 activity due to inhibitory autoantibodies is a key feature of acquired thrombotic thrombocytopenic purpura (TTP), leading to the persistence of ultralarge VWF multimers, platelet aggregation and disturbance of microcirculation. We followed 39 patients (8 male, 31 female, mean age 38 years) with clinical signs of TTP over a period between 5 days to 16 years and observed a total of 53 episodes of TTP. ADAMTS13 was measured with a collagen-binding assay and the FRETS-VWF73 based Technozym ADAMTS-13 assay (activity and antigen, respectively). ADAMTS13 inhibitor was measured with a modified Bethesda method with both the above mentioned assays, and with the Technozym ADAMTS-13 INH ELISA. Thirty-one patients had autoimmune TTP, and 47 episodes of TTP were analyzed in these patients. In all acute episodes, ADAMTS13 activity was below the detection limit (0.2 U/ml in 66% of the episodes (after median 160 days). In the remaining cases anti-ADAMTS13 antibodies persisted during remissions for up to 2 years. In 3 cases the antibody reoccurred after initial normalization of ADAMTS13 activity, and clinical relapses followed. In total, 21 relapses were observed after a median of 46 months (range 1– 87), all associated with low ADAMTS13 levels. Rituximab was given in 7 cases of relapsing TTP and resulted in complete, durable clearance of the antibodies in 100%. Determination of ADAMTS13-related parameters is useful to distinguish between autoimmune, hereditary, and secondary forms of TTP and to choose an appropriate therapy. It is also useful to predict the risk of relapse in patients with TTP in remission.

Beschreibung: Myeloproliferative diseases (MPDs) represent the commonest cause of splanchnic vein thrombosis (SVT), including Budd-Chiari syndrome (BCS) and portal vein thrombosis (PVT), but their diagnosis is hampered by changes secondary to portal hypertension, while their influence in the outcome of SVT remains unclear. We assessed the diagnostic and prognostic value of JAK2 and MPL515 mutations in 241 SVT patients (104 BCS, 137 PVT). JAK2V617F was found in 45% of BCS and 34% of PVT, while JAK2 exon 12 and MPL515 mutations were not detected. JAK2V617F was found in 96.5% of patients with bone marrow (BM) changes specific for MPD and endogenous erythoid colonies, but also in 58% of those with only one feature and in 7% of those with neither feature. Stratifying MPD diagnosis first on JAK2V617F detection would have avoided BM investigations in 40% of the patients. In BCS, presence of MPD carried significantly poorer baseline prognostic features, required hepatic decompression procedures earlier, but had no impact on 5-year survival. Our results suggest that JAK2V617F testing should replace BM investigations as initial test for MPD in patients with SVT. Underlying MPD is associated with severe forms of BCS, but current therapy appears to offset deleterious effects of MPD on the medium-term outcome.

Beschreibung: Autologous stem cell transplantation (ASCT) remains the standard consolidation therapy for patients with multiple myeloma (MM) and chemosensitive relapsed lymphoma (r-Ly). Peripheral blood as a source of stem cells (PBSC) has largely replaced marrow and has the advantage of improved engraftment rates. PBSC are routinely collected following administration of chemotherapy in combination with GCSF. However, the resultant pancytopenia poses a significant risk to patients and additional chemotherapy prior to ASCT may lead to increased end organ damage potentially precluding future therapies (including ASCT). Novel agents can achieve PBSC mobilisation without the use of cytotoxics. In the advent of such drugs, we reviewed the efficacy of, and complications experienced by patients during PBSC mobilisation. We also analysed the cost implications of adverse events. Of 151 consecutive attempts, 13.2% of patients failed to reach our criteria in order to attempt pheresis (1 × 104 CD34 cells/ml). Of those achieving target and undergoing pheresis, 6% did not achieve an adequate cell dose for future ASCT (2 × 106CD34+cells/kg) giving an overall failure rate of 19.2%. Furthermore 17.9% failed to harvest our ideal of 4 × 106/kg (permitting 〉1 ASCT procedure). Factors contributing to failure in achieving target CD34+ve PB count on univariate analysis were; 〉2 lines of previous chemotherapy and occurrence of neutropenic sepsis (NS (p=0.002, and 0.005 respectively). These factors remained significant on multivariate analysis (RR: 4.4 and 6.2). These same factors also affected CD34+ cell yield on both univariate and multivariate analysis (RR: 3.3 and 4.6). No differences were seen between MM and r-Ly. Overall, the complication rate was 34.4%, with 24.1% of patients suffering NS requiring admission. The mortality rate was 1.3% (NS and intra-cranial bleed). Of those developing NS, only 52% eventually harvested sufficient cells, but with a median delay of 3 days. The median cost of PBSC collection was $17,381.46 ($1,978.97–$39,355.73). NS significantly increased the cost of mobilisation at a median cost of $25,532.95 vs $16,4921) (p=

Beschreibung: Resolvin E1 (RvE1) is an omega-3 eicosapentaenoic acid (EPA)–derived lipid mediator generated during resolution of inflammation and in human vasculature via leukocyte-endothelial cell interactions. RvE1 possesses anti-inflammatory and proresolving actions. Here, we report that RvE1 in human whole blood rapidly regulates leukocyte expression of adhesion molecules. RvE1 in the 10- to 100-nM range stimulated L-selectin shedding, while reducing CD18 expression in both neutrophils and monocytes. When added to whole blood, RvE1 did not stimulate reactive oxygen species by either neutrophils or monocytes, nor did it directly stimulate cytokine/chemokine production in heparinized blood. Intravital microscopy (IVM) demonstrated that RvE1 rapidly reduced leukocyte rolling (∼ 40%) in venules of mice. In human platelet-rich plasma (PRP), RvE1 selectively blocked both ADP-stimulated and thromboxane receptor agonist U46619-stimulated platelet aggregation in a concentration-dependent manner. In contrast, Δ6,14-trans-RvE1 isomer was inactive. RvE1 did not block collagen-stimulated aggregation, and regulation of ADP-induced platelet aggregation was not further enhanced with aspirin treatment. These results indicate RvE1 is a potent modulator of leukocytes as well as selective platelet responses in blood and PRP, respectively. Moreover, the results demonstrate novel agonist-specific antiplatelet actions of RvE1 that are potent and may underlie some of the beneficial actions of EPA in humans.

Beschreibung: Central venous catheters (CVCs) are widely used in patients with hemato-oncological disease for indications such as administration of chemotherapy, parenteral nutrition, blood products and monitoring of hemodynamics. CVCs are a major source of hospital-acquired infection. The National Nosocomial Infections Surveillance System reported a rate of catheter-related bloodstream infection (CRBI) of five per 1000 central catheter-days. This study was a randomized trial in which patients with nontunneled catheters were randomly assigned to heparin-coated (7 French, polyurethane, double lumen; Arrow®, USA) or chlorhexidine and silver sulfadiazine impregnated CVCs (7 French, polyurethane, double lumen; Arrow®, USA). All CVCs were placed in the subclavian vein in the operating room. CRBI was defined according to Infectious Disease Society of America guidelines. The principal investigator determined whether infections were catheter related and had no knowledge of “the assigned arm” at the time of adjudication of the reference standard definition. Before catheter insertion, laboratory prothrombotic markers included factor V Leiden, the prothrombin gene Gly20210A mutation, plasma antithrombin levels, and protein C and S activity. In our study, all patients were systematically examined by ultrasonography just before, or less than 24 hours after, catheter removal and in case of clinical signs of thrombosis. Two radiologists performed the ultrasonography and were unaware of the allocation of the patients. Two hundred and twenty patients were randomly assigned. Eight patients were excluded after assignment. Ultimately, 212 patients were analyzed [median age: 28 years (7–60); 97 female and 115 male]. CRBI occurred in 6.7% (7/104) of those in the heparin-coated group (2.7 events per 1000 days) and in 4.6% (5/108) in the chlorhexidine and silver sulfadiazine group (2 events per 1000 days) (P=0.5). The microorganisms involved in CRBIs were coagulase-negative Staphylococcus (five cases in the heparin group and 3 cases in the antiseptic group), Staphylococcus aureus (one case in the heparin group), Candida parapsilosis (one case in the antiseptic group), Pseudomonas aeruginosa (one case in the heparin group and one case in the antiseptic group). We observed a CVC-related thrombosis in 11 patients (5.2%; 5 in the heparin group and 6 in the antiseptic group P=0.8). Catheter-related thrombosis and CRBI coincided in two patients and were not significantly correlated (P=0.6). Four (1.9%) and three (1.4%) patients had evidence of protein C and protein S deficiency, respectively. Only one patient had an antithrombin deficiency (0.5%). In total, 9 patients (4.2%) were heterozygous for the factor V Leiden mutation. Thrombosis was diagnosed in three out of 17 patients (17.6%) with a inherited prothrombotic abnormality compared to 8 out of 195 patients (4.1%) who did not have a thrombophilic marker (relative risk 4.3 CI 95% 1.2–14.7). Six and five patients experienced severe bleeding in the heparin and antiseptic groups, respectively. We did not observe heparin-induced thrombocytopenia and anaphylaxis to chlorhexidine was not reported. To our knowledge, this is the first randomized study comparing in haematological patients heparin-coated with antiseptic impregnated CVCs. Furthermore, our results suggest that inherited prothrombotic abnormalities contribute substantially to CVC-related thrombosis in hemato-oncological patients. Presently, the best options for reducing CRBI are heparin-coated and antibiotic-impregnated CVCs. A large, well designed randomized controlled trial is required to determine which of these is most effective.

Beschreibung: Tumor growth is associated with aberrant myelopoiesis, including the accumulation of CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs) that have the potential to promote tumor growth. However, the identity, growth, and migration of tumor-associated MDSCs remain undefined. We demonstrate herein that MDSCs at tumor site were composed primarily of bone marrow-derived CD11b+Gr-1hiLy-6Cint neutrophils and CD11b+Gr-1int/dullLy-6Chi macrophages. Unexpectedly, in vivo bromodeoxyuridine (BrdU) labeling and parabiosis experiments revealed that tumor-infiltrating macrophages were replenished more rapidly than neutrophils. CCR2 deficiency caused striking conversion of infiltrating cellular dominance from macrophages to neutrophils in the tumor with the excessive production of CXCR2 ligands and granulocyte-colony stimulating factor in the tumor without affecting tumor growth. Overall, our data established the identity and dynamics of MDSCs in a tumor-bearing host mediated by chemokines and elucidated unexpected effects of the paucity of macrophages on tumor development.

Beschreibung: Various human disorders are associated with misdistribution of iron within or across cells. Friedreich ataxia (FRDA), a deficiency in the mitochondrial iron-chaperone frataxin, results in defective use of iron and its misdistribution between mitochondria and cytosol. We assessed the possibility of functionally correcting the cellular properties affected by frataxin deficiency with a siderophore capable of relocating iron and facilitating its metabolic use. Adding the chelator deferiprone at clinical concentrations to inducibly frataxin-deficient HEK-293 cells resulted in chelation of mitochondrial labile iron involved in oxidative stress and in reactivation of iron-depleted aconitase. These led to (1) restoration of impaired mitochondrial membrane and redox potentials, (2) increased adenosine triphosphate production and oxygen consumption, and (3) attenuation of mitochondrial DNA damage and reversal of hypersensitivity to staurosporine-induced apoptosis. Permeant chelators of higher affinity than deferiprone were not as efficient in restoring affected functions. Thus, although iron chelation might protect cells from iron toxicity, rendering the chelated iron bioavailable might underlie the capacity of deferiprone to restore cell functions affected by frataxin deficiency, as also observed in FRDA patients. The siderophore-like properties of deferiprone provide a rational basis for treating diseases of iron misdistribution, such as FRDA, anemia of chronic disease, and X-linked sideroblastic anemia with ataxia.

Beschreibung: The JAK2V617F somatic point mutation has been described in patients with myeloproliferative disorders (MPDs). Despite this progress, it remains unknown how a single JAK2 mutation causes 3 different MPD phenotypes, polycythemia vera (PV), essential thrombocythemia, and primitive myelofibrosis (PMF). Using an in vivo xenotransplantation assay in nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice, we tested whether disease heterogeneity was associated with quantitative or qualitative differences in the hematopoietic stem cell (HSC) compartment. We show that the HSC compartment of PV and PMF patients contains JAK2V617F-positive long-term, multipotent, and self-renewing cells. However, the proportion of JAK2V617F and JAK2 wild-type SCID repopulating cells was dramatically different in these diseases, without major modifications of the self-renewal and proliferation capacities for JAK2V617F SCID repopulating cells. These experiments provide new insights into the pathogenesis of JAK2V617F MPD and demonstrate that a JAK2 inhibitor needs to target the HSC compartment for optimal disease control in classical MPD.

Beschreibung: The RECORD program comprised four pivotal phase III trials comparing the oral direct Factor Xa inhibitor, rivaroxaban, with subcutaneous enoxaparin for the prevention of venous thromboembolism (VTE) after total hip and knee replacement (THR and TKR). A total of 12,729 patients were randomized to receive rivaroxaban 10 mg once daily (od) starting 6–8 hours after surgery, or enoxaparin 40 mg od (RECORD1–3) starting the evening before surgery, or 30 mg every 12 hours (RECORD4) starting 12–24 hours after surgery. In RECORD1, patients undergoing THR received extended prophylaxis for 31–39 days. In RECORD2, patients undergoing THR received rivaroxaban for 31–39 days, or enoxaparin for 12±2 days followed by placebo up to 35 days. Patients undergoing TKR in RECORD3 and 4 received prophylaxis for 12±2 days. In all four trials the rivaroxaban regimens showed superior efficacy to the enoxaparin regimens with no significant increase in bleeding. The RECORD1–4 pooled analysis showed that rivaroxaban was significantly superior to enoxaparin for the primary endpoint, symptomatic VTE and all-cause mortality (0.8% vs 1.6%, respectively; p70–90, 〉90kg), gender, or renal function (CrCl 〉80, 50–80, 80 and 30 ml/min CrCl) groups studied. Similar trends were seen in the smaller subgroups of body weight extremes (〉110 kg subgroup - total VTE: 5/100 [5.0%] vs 8/119 [6.7%], respectively; ≤50 kg subgroup - any bleeding: 12/147 [8.2%] vs 8/162 [4.9%], respectively). These results suggest that age, body weight, gender and renal function (〉30 ml/min CrCl) have no clinically relevant effect on the efficacy or safety of rivaroxaban as thrombroprophylaxis following major orthopedic surgery. Subgroup Total VTE Odds ratio (rivaroxaban vs enoxaparin regimens) (95% CI Major VTE Odds ratio (rivaroxaban vs enoxaparin regimens) (95% CI) Any major or non-major bleeding Hazard ratio (rivaroxaban vs enoxaparin regimens) (95% CI) *Because of study exclusion criteria, few patients had CrCl 70–90kg 0.45 (0.34–0.59) 0.21 (0.11–0.39) 0.95 (0.77–1.17) 〉90kg 0.38 (0.25–0.57) 0.32 (0.13–0.69) 1.22 (0.95–1.58) Gender Male 0.35 (0.25–0.48) 0.21 (0.10–0.40) 1.12 (0.93–1.36) Female 0.47 (0.37–0.59) 0.27 (0.16–0.45) 1.06 (0.87–1.28) CrCl (ml/min) 〉80 0.35 (0.27–0.46) 0.22 (0.12–0.38) 1.12 (0.94–1.35) 50–80 0.60 (0.45–0.81) 0.30 (0.15–0.56) 0.99 (0.79–1.24)

Beschreibung: Transcription factors known as CCAAT enhancer binding proteins (C/EBPs) are involved in hematopoietic differentiation, including myelopoiesis and granulopoiesis. C/EBPβ-deficient mice develop normally; however, they exhibit defective macrophage function, resulting in increased susceptibility to infection. Little is known about the role of C/EBPβ in granulopoiesis; therefore, we examined granulopoiesis in C/EBPβ-deficient mice. Morphology, the number of peripheral blood and bone marrow cells, and the expression of genes specific for the myeloid lineage were normal in C/EBPβ-deficient mice. Interestingly, the hematopoietic progenitor cells of C/EBPβ-deficient mice did not respond normally to granulocyte/macrophage-colony stimulating factor and granulocyte colony stimulating factor. In addition, C/EBPβ-deficient neutrophils displayed enhanced apoptosis compared with wild-type neutrophils. Our present results indicate that C/EBPβ helps regulate survival of neutrophils, downstream of the granulocyte colony stimulating factor receptor.

Beschreibung: The coagulation system is activated in sickle cell disease (SCD) and acute vaso-occlusion may heighten hypercoagulability. Protein C, a natural anticoagulant, has been reported to be low in individuals with SCD. Therefore, the natural anticoagulation pathway may be disrupted in SCD. The objective of this study is to more fully evaluate the protein C pathway in murine and human SCD by examining levels of: coagulation activation; protein C activity; thrombomodulin (TM); and endothelial protein C receptor (EPCR). In order to assess the level of activation of the coagulation system, we measured plasma thrombin/antithrombin (TAT) complex levels in humans and mice. TAT levels were elevated in 22 humans with SCD versus 9 healthy controls at baseline, and levels increased further in 15 individuals with SCD during acute vaso-occlusive events (5.6±1.2 vs. 2.4±0.2 vs. 9.2±1.8ug/L respectively, p=0.02). In order to study acute vaso-occlusive events in mice, we developed a model of acute vaso-occlusion by exposing Berkeley SCD mice to 3 hours of hypoxia (FI02 8–10%) followed by 2, 4, or 21 hours of reoxygenation in room air (HR2, HR4, HR21). In support of our human findings, TAT was elevated in SCD mice compared to HbA mice at baseline, and increased further in SCD mice exposed to HR2 (n=5–14 per group, p

Beschreibung: The addition of Rituximab (R) to front-line combination chemotherapy has substantially improved the long term perspectives of patients with advanced stage follicular lymphoma (FL) resulting in median progression free survival (PFS) times of more than 36 months. In this situation the potential role of myeloablative herapy with subsequent autologous stem cell transplantation (ASCT) in 1st remission needs to be critically reassessed. For this purpose two consecutive studies of the GLSG were analyzed both randomizing in 1st remission for Interferon alpha (IFN) maintenance therapy versus ASCT. In the GLSG trial ‘96 initial therapy comprised a randomized comparison of CHOP versus Mitoxantrone, Chlorambucil and Prednisone (MCP) (total patient number 312) while in GLSG study ‘00 CHOP was randomly compared to R-CHOP (total patient number 268). Hence, both studies had an identical treatment arm of CHOP followed by either IFN maintenance or ASCT. In both studies, the identical treatments revealed superimposable results with a 5 years PFS of 27% after CHOP and IFN maintenance and 66% after CHOP followed by ASCT. Taking these data as internal control R-CHOP followed by IFN maintenance achieved a 5 year PFS of 67% which is hence comparable to CHOP followed by ASCT. R-CHOP followed by ASCT, however, revealed a 5 year PFS of 79% with only one relapse after 24 months. Figure Figure Although the difference between R-CHOP followed by IFN maintenance or ASCT is currently not yet significant it strongly suggests a beneficial effect of ASCT in the era of R-CHOP front-line therapy particularly for intermediate of high risk patients with advanced stage FL.

Beschreibung: Hemojuvelin (HJV) is a coreceptor for bone morphogenetic protein (BMP) signaling that regulates hepcidin expression and iron metabolism. However, the precise combinations of BMP ligands and receptors used by HJV remain unknown. HJV has also been demonstrated to bind to neogenin, but it is not known whether this interaction has a role in regulating hepcidin expression. In the present study, we show that BMP-2, BMP-4, and BMP-6 are endogenous ligands for HJV in hepatoma-derived cell lines, and that all 3 of these ligands are expressed in human liver. We demonstrate in vitro that HJV selectively uses the BMP type II receptors ActRIIA and BMPRII, but not ActRIIB, and HJV enhances utilization of ActRIIA by BMP-2 and BMP-4. Interestingly, ActRIIA is the predominant BMP type II receptor expressed in human liver. While HJV can use all 3 BMP type I receptors (ALK2, ALK3, and ALK6) in vitro, only ALK2 and ALK3 are detected in human liver. Finally, we show that HJV-induced BMP signaling and hepcidin expression are not altered by neogenin overexpression or by inhibition of endogenous neogenin expression. Thus, HJV-mediated BMP signaling and hepcidin regulation occur via a distinct subset of BMP ligands and BMP receptors, independently of neogenin.

Beschreibung: Multiple myeloma (MM) is characterized by osteolytic bone lesions (OBL) that arise as a consequence of osteoblast inactivation and osteoclast activation adjacent to tumor foci within bone. Wnt signaling in osteoblasts regulates osteoclastogenesis through the differential activation and inactivation of Receptor Activator of Nuclear factor Kappa B Ligand (RANKL) and osteoprotegerin (OPG), positive and negative regulators of osteoclast differentiation, respectively. We demonstrate here that MM cell–derived DKK1, a soluble inhibitor of canonical Wnt signaling, disrupted Wnt3a-regulated OPG and RANKL expression in osteoblasts. Confirmed in multiple independent assays, we show that pretreatment with rDKK1 completely abolished Wnt3a-induced OPG mRNA and protein production by mouse and human osteoblasts. In addition, we show that Wnt3a-induced OPG expression was diminished in osteoblasts cocultured with a DKK1-expressing MM cell line or primary MM cells. Finally, we show that bone marrow sera from 21 MM patients significantly suppressed Wnt3a-induced OPG expression and enhanced RANKL expression in osteoblasts in a DKK1-dependent manner. These results suggest that DKK1 may play a key role in the development of MM-associated OBL by directly interrupting Wnt-regulated differentiation of osteoblasts and indirectly increasing osteoclastogenesis via a DKK1-mediated increase in RANKL-to-OPG ratios.

Beschreibung: Various pathologies are characterized by the accumulation of toxic iron in cell compartments. In anemia of chronic disease, iron is withheld by macrophages, leaving extracellular fluids iron-depleted. In Friedreich ataxia, iron levels rise in the mitochondria of excitable cells but decrease in the cytosol. We explored the possibility of using deferiprone, a membrane-permeant iron chelator in clinical use, to capture labile iron accumulated in specific organelles of cardiomyocytes and macrophages and convey it to other locations for physiologic reuse. Deferiprone's capacity for shuttling iron between cellular organelles was assessed with organelle-targeted fluorescent iron sensors in conjunction with time-lapse fluorescence microscopy imaging. Deferiprone facilitated transfer of iron from extracellular media into nuclei and mitochondria, from nuclei to mitochondria, from endosomes to nuclei, and from intracellular compartments to extracellular apotransferrin. Furthermore, it mobilized iron from iron-loaded cells and donated it to preerythroid cells for hemoglobin synthesis, both in the presence and in the absence of transferrin. These unique properties of deferiprone underlie mechanistically its capacity to alleviate iron accumulation in dentate nuclei of Friedreich ataxia patients and to donate tissue-chelated iron to plasma transferrin in thalassemia intermedia patients. Deferiprone's shuttling properties could be exploited clinically for treating diseases involving regional iron accumulation.

Beschreibung: Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in patients treated for X-linked severe combined immunodeficiency. Here, we analyzed whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst case scenario,” we transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of gamma retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1, or ΔTrkA, a constitutively active mutant of TrkA. After transplantation into RAG-1–deficient recipients (Ly5.2), animals that received stem cell transplants developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated polymerase chain reaction analysis revealed monoclonality or oligoclonality of the malignancies. In striking contrast, none of the mice that received T-cell transplants transduced with the same vectors developed leukemia/lymphoma despite persistence of gene-modified cells. Thus, our data provide direct evidence that mature T cells are less prone to transformation than hematopoietic progenitor cells.

Beschreibung: In this randomized multicenter study of 136 patients, 6 courses of CHOP (cyclo-phosphamide/doxorubicin/vincristine/prednisone) followed by rituximab (CHOP-R) were compared with rituximab-supplemented high-dose sequential chemotherapy with autografting (R-HDS) to assess the value of intensified chemo-therapy as a first-line treatment for high-risk follicular lymphoma (FL) after the introduction of monoclonal antibodies. The analysis was intention to treat with event-free survival (EFS) as the primary endpoint. Complete remission (CR) was 62% with CHOP-R and 85% with R-HDS (P 〈 .001). At a median follow-up (MFU) of 51 months, the 4-year EFS was 28% and 61%, respectively (P 〈 .001), with no difference in overall survival (OS). Molecular remission (MR) was achieved in 44% of CHOP-R and 80% of R-HDS patients (P 〈 .001), and was the strongest independent outcome predictor. Patients relapsing after CHOP-R underwent salvage R-HDS in 71% of cases. Salvage R-HDS had an 85% CR rate and a 68% 3-year EFS (MFU, 30 months). We conclude that (1) achieving MR is critical for effective disease control, regardless of which treatment is used; (2) R-HDS ensures superior disease control and molecular outcome than CHOP-R, but no OS improvement; and (3) CHOP-R failures have a good outcome after salvage R-HDS, suggesting that relapsed/refractory FL could be the most appropriate setting for R-HDS–like treatments. This trial was registered at www.clinicaltrials.gov as no. NCT00435955.

Beschreibung: Functional characterization of signaling pathways that critically control mantle cell lymphoma (MCL) cell growth and survival is relevant to designing new therapies for this lymphoma. We herein demonstrate that the constitutive activation of Akt correlates with the expression of the phosphorylated, inactive form of PTEN. Phosphatidyl-inositol-3 kinase (PI3-K)/Akt or mammalian target of rapamycin (mTOR) inhibition decreased the growth of both primary MCL cultures and established cell lines and antagonizes the growth-promoting activity of CD40 triggering and IL-4. These effects are mediated by nuclear accumulation of the p27Kip1 inhibitor induced by down-regulation of the p45Skp2 and Cks1 proteins, which target p27Kip1 for degradation. Moreover, Akt inhibition down-regulated cyclin D1 by promoting its proteasome-dependent degradation driven by GSK-3. Intriguingly, mTOR inhibition affected cyclin D1 proteolysis only in MCL cells in which GSK-3 is under the direct control of mTOR, suggesting that different MCL subsets could be differently responsive to mTOR inhibition. Finally, PI3-K/Akt inhibitors, but not rapamycin, induced variable levels of caspase-dependent apoptosis and reduced telomerase activity. These results indicate that Akt and mTOR activation have distinct functional relevance in MCL and suggest that targeting Akt may result in more effective therapeutic effects compared with mTOR inhibition.

Beschreibung: MRC UKALLXII/ECOG2993 accrued 739 ECOG pts over 13 years. ECOG’s reference laboratories centrally characterized 613 (91%) and 505 were B-Lin ALLs. Expression of 32 antigens (Ags), including 9 myeloid-associated Ags, was determined by multiparameter flow cytometry on gated blasts, both as percentage of Ag expressing blasts and intensity of antibody staining (equivalent of Ag density). To test for the potential therapeutic efficacy of monoclonal antibodies, such as rituximab, epratuzumab, alemtuzumab, or gemtuzumab in future trials, expression of Ags for these antibodies (CD20, CD22, CD52, or CD33, respectively) was determined. In E2993, 14% of pts typed as Pro-B (CD10 negative), 71% as Early Pre-B (CD10 positive), 13% as Pre-B (intracytoplasmic mu positive), and 2% as Mature B-ALL (surface mu chains positive). CD20 expression correlated with the stage of B-lymphoid maturation (p=5.4E-13) (see table). Median CD20 intensity of fluorescence was lowest in Pro-B and highest in Mature B-ALL. Membrane expression of CD22 was lower in Pro-B compared with the other three groups, Early Pre-B, Pre-B and Mature B (p=1.0E-5) but indistinguishable among the latter three (p=0.21). While levels of CD20 and CD22 had no effect on complete remission (CR) and overall survival (OS) univariatly, higher CD22 expression was significantly correlated with better OS (p=0.02) in a bivariate Cox model of CD20 and CD22. With respect to myeloid Ags, CD33/CD13 expression was associated with Early Pre-B (p=0.02), and CD65(s)/CD15(s) with Pro-B ALL (p

Beschreibung: INTRODUCTION: Eltrombopag (PROMACTA®/REVOLADE®; GlaxoSmithKline, Collegeville, PA, USA) is a first-in-class, oral, small molecule, non-peptide, thrombopoietin receptor agonist being studied for the treatment of thrombocytopenia related to a variety of conditions. METHODS: RAISE was a 6-month, randomized, double-blind, placebo-controlled, phase III study that evaluated the efficacy and safety of eltrombopag in previously treated adults with chronic idiopathic thrombocytopenic purpura (ITP) with platelet counts 15% had received at least 3 prior ITP medications. Patients who received eltrombopag were 8 times more likely to achieve platelet counts 50,000 to 400,000/μL during the 6-month treatment period compared with patients on placebo (OR [95% CI] = 8.2 [4.32, 15.38]; P

Beschreibung: T-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3+CD8+ cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P5 is the predominant S1P receptor in leukemic LGLs, whereas S1P1 is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

Beschreibung: Activation of human plasmacytoid dendritic cells (pDCs) with ligands for Toll-like receptors (TLRs) 7 and 9 induces the secretion of type I interferons and other inflammatory cytokines as well as pDC differentiation. Transcripts for 2 members of the CD300 gene family, CD300a and CD300c, were identified on pDCs during gene expression studies to identify new immunoregulatory molecules on pDCs. We therefore investigated the expression of CD300a and CD300c and their potential regulation of pDC function. CD300a/c RNA and surface expression were downregulated after stimulation of pDCs with TLR7 and TLR9 ligands. Exogenous interferon (IFN)-α down-regulated CD300a/c expression, whereas neutralizing IFN-α abolished TLR ligand–induced CD300a/c down-regulation. This implicates IFN-α in regulating CD300a/c expression in pDCs. In addition, IFN-α favored tumor necrosis factor (TNF)-α secretion by CpG-induced pDCs. CD300a/c triggering by cross-linking antibody reduced TNF-α and increased IFN-α secretion by pDCs. Furthermore, CD300a/c triggering, in the presence of neutralizing IFN-α, further reduced TNF-α secretion. These data indicate that CD300a and CD300c play an important role in the cross-regulation of TNF-α and IFN-α secretion from pDCs.

Beschreibung: Receptor or nonreceptor tyrosine kinases (TKs) are known to play an important role in leukemogenesis. Here we studied the level of protein tyrosine phosphorylations in a series of fresh AML samples and evaluated the effect of TK inhibitors. Compared with normal hematopoietic progenitors, a high level of tyrosine phosphorylation was detected in most acute myeloid leukemia (AML) samples. The Src family kinases (SFKs) appeared constitutively activated in most cases, including in the CD34+CD38−CD123+ compartment as revealed by the level of phosphorylated tyrosine 416. Lyn was the major SFK family member expressed in an active form in AML cells where it was abnormally distributed throughout the plasma membrane and the cytosol as opposed to normal hematopoietic progenitors. The SFK inhibitor, PP2, strongly reduced the global level of tyrosine phosphorylations, inhibited cell proliferation, and induced apoptosis in patient samples without affecting normal granulomonocytic colony forming units. Moreover, silencing Lyn expression by small interfering RNA in primary AML cells strongly inhibited proliferation. Interestingly, a link between Lyn and the mTOR pathway was observed as PP2 and a Lyn knockdown both affected the phosphorylation of mTOR targets without inhibiting Akt phosphorylation. Lyn should be considered as a novel pharmacologic target for AML therapy.

Beschreibung: Background. In 2005 we analyzed the results of a prospective randomized phase III intergroup trial evaluating the role of rituximab (R) both in remission induction and maintenance treatment of 465 relapsed/resistant follicular lymphoma (FL) patients. Major conclusions were that addition of R to CHOP induction yielded an increased ORR and CR rate, and that R maintenance strongly improved median progression free survival (PFS), both after induction with CHOP and R-CHOP, and overall survival (OS (van Oers et al Blood2006;108:3295). At that time the median follow for the maintenance phase was 33 months. Now we report the long-term outcome of maintenance treatment, with a median follow up of 6 years from start of maintenance. Study design. Patients with stages III or IV FL at initial diagnosis and relapsed after or resistant to a maximum of two non-anthracycline containing systemic chemotherapy regimens, were randomized to remission induction with either 6 cycles of standard CHOP (once every 3 weeks) or CHOP + R (375 mg/m2 at day 1 of each cycle of CHOP). Those with a complete or partial remission after 6 cycles of therapy underwent a second randomization to no further treatment (observation) or maintenance treatment with R (375 mg/m2 once every 3 months) until relapse or for a maximum period of two years. Results. 465 patients were randomized to induction with either CHOP (231) or R-CHOP (234). As reported, CHOP and R-CHOP induction yielded similar partial response rates (57% vs.56%), but significantly different CR rates (16% and 29%; p=0.0001). 334 patients were randomized to either R maintenance treatment (167) or observation (167). R maintenance resulted in a highly significant improvement of PFS: median 3.7 years versus 1.3 years in the observation arm (p

Beschreibung: Hematopoietic stem cell (HSC) homeostasis and self-renewal are regulated by intrinsic cytokine signaling pathways. One important signaling axis for HSC is the cell surface receptor, Mpl, and its ligand, thrombopoietin (Tpo). Upon Tpo stimulation, Mpl activates Janus Kinase (JAK2) that triggers a cascade of downstream signal transduction pathways that regulates many aspects of cell development. Under steady-state conditions, mice lacking the inhibitory adaptor protein Lnk harbor an expanded HSC pool with enhanced self-renewal. Surprisingly, we found that Lnk−/− HSCs have an increased quiescent fraction, decelerated cell cycle kinetics, and enhanced resistance to repeated 5-Florouracil (5-FU) treatments in vivo compared to wild type HSCs. We further provided genetic evidence demonstrating that Lnk controls HSC quiescence and self-renewal predominantly through Mpl. Consistent with this observation, Lnk deficiency in HSCs potentiates JAK2 activation in response to TPO. Biochemical experiments reveal that Lnk directly binds to phosphorylated tyrosine residues in JAK2 following TPO stimulation. Dysregulation of cytokine receptor signaling pathways leads to hematological malignancies. Abnormal activation of JAK2 by a chromosomal translocation between the transcription factor Tel and JAK2 (Tel/JAK2) was shown to cause atypical Chronic Myelogenous Leukemia (aCML). Recently, the JAK2 V617F mutation has been observed at high frequencies in several myeloproliferative diseases (MPDs). The JAK2V617F mutant retains Lnk binding ability, suggesting Lnk status could modify MPD development. Indeed, we found that loss of Lnk accelerates oncogenic JAK2- induced CML/MPD in the mouse transplant models. Therefore, we identified Lnk as a physiological negative regulator of JAK2 in stem cells that may contribute to leukemic transformation conferred by oncogenic JAK2.

Beschreibung: Erythropoiesis is a developmentally important process, whereby multipotent hematopoietic stem cells differentiate into mature erythrocytes. Erythropoietin (EPO) is a critical regulator in this process and mediates its signal via the erythropoietin receptor (EPO-R) and the primary associated tyrosine kinase, JAK2. EPO, EPO-R and JAK2 play a crucial role in erythropoiesis, as deficiency in any of these proteins results in an embryonic lethal anemia. Structural-functional studies and murine knock-in models have shown EPO-R pTyr-343 to play a critical role in EPO mediated signalling. STAT-5 is activated by EPO-R pTyr-343, but STAT5ΔN mice do not have any profound erythroid abnormalities. Such evidence has led our group to hypothesize that other SH2 containing effectors interact with EPO-R pTyr-343. Cloning of Ligand Target screening was utilized to demonstrate that EPO-R pTyr-343 binds to adaptor protein SH2-Bβ. SH2-B contains multiple protein-protein interaction domains including multiple proline-rich regions, a PH domain and an SH2 domain. Although SH2-B does play a role in a number of signaling pathways, it is not required for embryonic development. Since SH2-B is a potent regulator of JAK2 in context of Growth Hormone and Leptin signaling, and it can directly interact with EPO-R pTyr-343, we hypothesize that SH2-B functions as an important adaptor protein downstream of the EPO-R.H2-B constitutively associates to the inactive EPO-R, an interaction that is independent of JAK2 binding to the EPO-R. Upon EPO stimulation, enhanced SH2-dependent binding of SH2-B to pTyr-343 and pTyr-401 of the EPO-R was confirmed utilizing a panel of EPO-R truncation mutants. The EPO mediated interaction between SH2-B and activated EPO-R is both dose and time dependent. EPO stimulation also results in SH2-B serine and threonine phosphorylation. Importantly, the interaction of SH2-B and EPO-R was observed in erythroid cell lines and primary murine splenocytes. The function of SH2-B in EPO signaling was investigated via knocking down SH2-B in Ba/F3-EPO-R cells. Knock down of SH2-B results in hypersensitive EPO-dependent phosphorylation of multiple targets including the EPO-R, JAK2, STAT5 and Erk1/2. It is evident that SH2-B is a global negative regulator of EPO-dependent signaling via its ability to affect EPO-dependent JAK2 activation.

Beschreibung: The 26S proteasome inhibitor bortezomib (BZ), which increases intracellular unfolded protein levels and toxicity through endoplasmic reticulum (ER) stress response, was shown to have a single agent activity in relapsed mantle cell lymphoma (MCL). Here we have determined that treatment with hydroxamic acid analogue (HA) pan-histone deacetylase (HDAC) inhibitor (HDI), e.g., panobinostat (LBH589, Novartis Pharmaceuticals Inc) induces the CDK inhibitors p21 and p27, and attenuates the levels of c-Myc, CDK4 and cyclin D1 in the cultured (Jeko-1, MO-2058 and Granta-519) and in primary patient-derived MCL cells. In a dose-dependent manner, panobinostat also induced Bax and Bak, and attenuated Bcl-xL, XIAP, survivin, AKT and c-Raf levels, resulting in growth inhibition and apoptosis of MCL cells. We have previously demonstrated that HDAC6 deacetylates heat shock protein (hsp) 90, as well as shuttles and sequesters misfolded and polyubiquitylated proteins into the protective perinuclear aggresome.. By inhibiting HDAC6, panobinostat (10 to 50 nM) induced acetylation of hsp90 in MCL cells. This inhibited the ATP binding and co-chaperone association, and abrogated the chaperone function of hsp90 for the MCL- relevant, hsp90 client proteins, e.g., cyclin D1, CDK4, c-Raf and AKT in the cultured and primary MCL cells. Panobinostat mediated inhibition of HDAC6 abrogated formation of the aggresome and augmented endoplasmic reticulum (ER)-based unfolded protein response (UPR). Treatment of MCL cells with BZ induced the formation of aggresome (as detected by confocal immuno-fluorescence microscopy and electron microscopy), as well as induced UPR and ER stress response. The latter was associated with BZ-mediated increased levels of GRP78, the spliced form of XBP1 (XBP1s) and p-eIF2α protein. As compared to the control siRNA treated cells, knockdown of GRP78 by siRNA markedly increased BZ-induced CHOP and Noxa levels and significantly augmented BZ-induced apoptosis of cultured MCL cells. Co-treatment of MCL cells with panobinostat abrogated BZ-induced aggresome formation, decreased the levels of ATF4, XBP1s and p-eIF2α, as well as increased the levels of CHOP, Noxa and GADD34. Ultrastructural analysis of Jeko-1 cells also revealed that co-treatment with panobinostat and BZ showed pronounced ER dilatation compared to panobinostat treatment alone, suggestive of enhanced ER stress. Higher and persistent CHOP and Noxa levels suggested a protracted ER-stress, associated with synergistic increase in apoptosis of MCL but not normal CD34+ bone marrow progenitor cells (p 〈 0.01). Conversely, knockdown of CHOP levels by siRNA significantly inhibited panobinostat and BZ-induced cell death of MCL cells. Results of ongoing in vivo studies of panobinostat and/or BZ in the NOD/SCID mouse xenograft model of Jeko-1 MCL cells will be presented. These findings strongly support further in vivo evaluation of the efficacy of the combination of panobinostat with BZ against human MCL. Additionally, the findings create the rationale to develop targeted knockdown of GRP78 as a novel strategy to augment lethal ER stress due to panobinostat and BZ and resulting activity against MCL cells.

Beschreibung: AML1-ETO is generated from t(8;21)(q22;q22), which is commonly associated with acute myeloid leukemia (AML), especially FAB M2 AML. While full length AML1-ETO fails to promote leukemia due to its detrimental effects on cell proliferation, an alternatively spliced isoform without its C-terminal NHR3 and NHR4 domains strongly induces leukemia. The NHR4 domain has a zinc-chelating topology and has been implicated in transcriptional repression through recruitment of the NCoR/SMRT complexes. Here, we report that disruption of the zinc-chelating structure in the NHR4 domain of AML1-ETO by replacing only one critical amino acid leads to rapid onset of leukemia, suggesting that the NHR4 domain is responsible for generating inhibitory effects on leukemia development. To identify the molecular mechanism of this leukemogenic effect, we searched for NHR4-interacting factors with yeast two hybrid screening, and identified a novel NHR4-interacting protein, SON, a potential DNA/RNA-binding protein. The N-terminal fragment of SON with 81 amino acids, which is encoded by exons 1 and 2, was sufficient to interact with the NHR4 domain of ETO. Interaction of endogenous SON and AML1-ETO was also detected in t(8;21) AML cell lines. A serine substitution of cysteine 663 which disrupts the zinc-chelating topology of the NHR4 domain completely abolished its interaction with SON, while retaining the interaction with another ETO-binding protein, N-CoR. Knockdown of SON by siRNA resulted in significant growth arrest. Disruption of the interaction between AML1-ETO and endogenous SON by overexpressing the SON N-terminal fragment rescued cells from AML1-ETO-induced growth arrest. These results suggest that SON is an indispensable factor for cell growth and the interaction between endogenous SON and AML1-ETO generates negative effects on cell growth. In t(8;21) AML patient-derived primary leukemic cells and cell lines, abnormal cytoplasmic localization of SON was detected, which may keep cells proliferating in the presence of full length AML1-ETO. Taken together, these results uncovered the crucial role of the NHR4 domain in determination of cellular fate during AML1-ETO-associated leukemogenesis and revealed the interaction of the NHR4 domain with SON, an essential protein for normal cell growth.

Beschreibung: The majority of patients with acute myelogenous leukemia (AML) who achieve complete remission (CR) with initial chemotherapy will eventually relapse. Most relapses occur in the first three years and rare patients relapse after being in CR for more than 5 years. We have reviewed retrospectively 2347 patients with AML treated at M D Anderson Cancer Center from January 1980 to July 2008. Of the total cohort, 1366 patients achieved CR and among these 942 patients relapsed. We identified 11 patients (1.16%) who relapsed after being in CR 〉5 years; these patients are the focus of this analysis. There were 4 females and 7 males. Their median age was 66 years (range 37–79), and their median presentation white blood cell (WBC) count was 2.3 x109/L (range, 1.1–92.3). The FAB classification was M2 in 5 patients, M1, M4, M5, M6 in 1 patient each, and unknown in 2 patients. Initial cytogenetics were diploid in 6, del(7q) in one, miscellaneous in 1, and unavailable in 3 patients. Initial therapy was with combination of idarubicin (Ida) and cytarabine (Ara-C) in 4 patients (1 with additional fludarabine), amsacrine based in 3, daunorubicin (Dauno) single agent in 2 and other agents in 2 patients. All patients except one achieved CR after the first induction course; one patient needed 2 cycles of induction to achieve CR. None underwent an allogeneic stem cell transplant in first CR. The median duration of CR was 81 months (range, 60–137). At the time of relapse, median WBC count was 4.4 x109/L (range 1.7–48.8). Karyotype at relapse was diploid in 2, del(5)del(7) in 1, del(6)del(7) in 1, trisomy 8 in 1, hyperdiploid in 2, add(2q) and add(6q) in 1 each and unavailable in 2 patients. The karyotype at relapse was different from the initial finding in 8 of 8 patients with available data at both time points. Treatment for relapse included Ida (or Dauno) with Ara-C in 8 patients (1 with additional fludarabine), and other agents in 3 patients. The response to treatment was CR in 4, partial remission in 2, resistant in 4 and unknown in 1. No patient underwent an allogeneic stem cell transplant in second CR. The median duration of the second CR was 2 months (range 0–37). The median survival after relapse was 6.4 months (range 1–39). Median survival from initial diagnosis of AML was 107 months (range 68–143). We conclude that late relapses in AML (〉5 years after CR) are infrequent (1.16% of all relapses) and response to their subsequent therapy is poor; best responses occur with a regimen similar to the initial induction regimen. The karyotype at relapse is frequently different raising the question of a second AML versus relapse with the original clone.

Beschreibung: Pegylated E. Coli Asparaginase (Peg-ASP) (Oncospar, Medac UK) 1000 units/m2 administered intra-muscularly replaced native E. Coli Asparaginase (Elspar, MSD) throughout treatment in the current UK trial for children and young adults with ALL, UKALL 2003. The trial opened in October 2003 to patients aged 1 – 21 years (increased to 25 years recently). Included in these analyses are the 1542 patients enrolled and followed up to 31/10/2007. Standard and Intermediate risk patients receive two doses Peg-ASP in induction at days 4 and 18 and a single dose on day 4 of each of two delayed intensifications. High risk patients receive an additional 8 doses during consolidation, interim maintenance and 2 delayed intensification courses. Although results of the randomised interventions remain blinded, overall event free survival (EFS) is significantly better for patients in UKALL 2003 compared with its predecessor, ALL 97/99 (3 year EFS: 92% (se 0.9) vs 87% (se 1.1) - p=0.005), particularly for patients in NCI high risk, T cell and slow early response categories. Both trials included risk stratification by NCI risk, blast karyotype, and morphological marrow response at day 8/15 of induction to allocate patients to one of 3 treatment regimens (A, B and C – escalating intensity from A to C). NCI standard risk (SR) patients received a 3 drug induction whilst high risk (SR) patients received 4 drugs including daunorubicin. Patients with a slow early response at day 8 (NCI HR) or day 15 (NCI SR) of induction were transferred to Regimen C. The only difference between the trials in treatment administered to all patients was the use of Peg-ASP and dexamethasone in UKALL 2003 instead of native E. Coli asparaginase and a dexamethasone vs prednisolone randomisation in ALL97/99. Only patients with overt CNS disease (CNS3) at presentation received cranial radiotherapy, and high dose Methotrexate was not part of the protocol in either trial. CNS directed therapy comprised 19 – 26 doses of intrathecal methotrexate given over 2 – 3 years depending on regimen and gender. Comparison of cohorts receiving otherwise similar treatment in the two trials suggests that Peg-ASP is an important contributor to the improved outcome seen in the high risk subgroups. In NCI HR patients, EFS is significantly better in UKALL2003 (3 year EFS: 88% (se 1.6)) compared with ALL97/99 patients who received Elspar and dexamethasone (79% (se 3.8)) (p=0.01). For T-cell patients, 3 year EFS in UKALL2003 is 86% (se 3.3), and 72% (se 9.0) in ALL97/99 (p=0.1). The incidence of Peg-ASP related serious toxicities has been relatively low: thrombosis 3% (CNS

Beschreibung: Off-patent drugs with previously unrecognized anti-cancer activity could be rapidly repurposed for this new indication given their prior testing for safety and toxicity. To identify such compounds, we developed, automated and conducted a cell-based chemical screen of 4800 off-patent drugs and chemicals. From this chemical screen, we identified the off-patent antimicrobial, ciclopirox olamine (CPX) that is currently used for the topical treatment of cutaneous fungal infections, but has not been previously evaluated as a systemic agent for the treatment of malignancy. As an anti-fungal agent, the mechanism of action of CPX is not well understood, but appears related to binding intracellular iron and inhibiting iron containing enzymes. To explore its efficacy and mechanism of action as an anticancer agent, leukemia, myeloma, and solid tumor cell lines were treated with increasing concentrations of CPX. 72 hours after incubation, cell viability was measured by the MTS assay. CPX decreased cell viability in 5/9 leukemia, 3/6 myeloma, and 3/5 solid tumor cells with an LD50 〈 5 uM, a concentration that is pharmacologically achievable based on prior animal studies investigating CPX as an anti-fungal. Cell death was confirmed by the presence of a subG1 peak by flow cytometry after staining cells with propidium iodide. In contrast, CPX was less toxic to MRC 5, LF1, and GMO 5757 non malignant fibroblasts with an LD50 〉 20 uM. Next, we evaluated CPX in combination with cytarabine and daunorubicin, standard chemotherapeutic agents used in the treatment of AML. In AML cell lines, CPX synergistically enhanced the cytotoxicity of cytarabine as determined by the median effect isobologram analysis. Specifically, the combination indices (CI) at the EC50, 75 and 90 were 0.18, 0.19, and 0.24, respectively, where a CI 〈 1 denotes synergy. In contrast, the addition of CPX to daunorubicin produced only additive effects. Given the effects in leukemia cells lines, we evaluated the effects of oral CPX in 3 mouse models of leukemia. Sublethally irradiated NOD-SCID mice were injected subcutaneously with OCI-AML2 or K562 human leukemia cells or intraperitoneally with MDAY-D2 murine leukemia cells. After tumor implantation, mice were treated with CPX (25mg/kg) in water or water alone by oral gavage. Oral CPX decreased tumor weight and volume in all 3 mouse models by up to 65% compared to control without evidence of weight loss or gross organ toxicity. Mechanistically, CPX arrested cells in the G1/S phase of the cell cycle and downregulated the expression of survivin, Cyclin D1, and the transcription factors YY1 and FTII-D prior to the onset of cell death. Consistent with effects as an anti-fungal, CPX bound intracellular iron in the malignant cells and its ability to bind intracellular iron was functionally important for its cytotoxicity. In contrast to CPX, deferoxamine, a more avid extracellular iron chelator, was not significantly cytotoxic with an IC25 〉 10uM. The highest demand for intracellular iron occurs during the late G1 and S phases due, in part, to the activity of the iron-requiring enzyme ribonucleotide reductase. Therefore, we examined the effects of CPX on the activity of ribonucleotide reductase. By electron paramagnetic resonance (EPR), CPX inhibited ribonucleotide reductase at concentrations associated with cell death. Cell lines resistant to CPX-mediated inhibition of ribonucleotide reductase were also resistant to CPX-induced cell death, supporting a mechanism of action linked to ribonucleotide reductase. Thus, in summary, the off-patent anti-fungal agent CPX induces cell death through its ability to bind intracellular iron. Its ability to inhibit the iron-containing enzyme ribonucleotide reductase appears functionally important for its mechanism of action. CPX displays previously unrecognized anti-cancer activity at concentrations that are pharmacologically achievable. Thus, CPX could be rapidly repurposed for the treatment of malignancies including leukemia and myeloma.

Beschreibung: Background: Patients (pts) with sAML are evaluated together in large AML trials regardless of whether their sAML arises from antecedent MDS vs. from MPD vs. t-AML. Prognostic factors and outcomes may differ among these subgroups, and a prognostic scoring system would be helpful in determining which patients would benefit from induction chemotherapy. Methods: We conducted a retrospective review of all pts with newly diagnosed, pathologically-confirmed AML at Cleveland Clinic between 1997 and 2007 to identify sAML pts treated with cytarabine-based induction chemotherapy. Data on known prognostic factors (age, white blood cell count (WBC) at diagnosis, cytogenetic risk groups (as defined by CALGB 8461), and AML etiology) were collected as baseline characteristics and controlled for in stepwise multivariable analyses. Complete response (CR) and overall survival (OS) were analyzed. A prognostic scoring system for OS was developed based on the number of poor prognostic features present, derived from significant multivariable factors. Pts received 1 point for adverse cytogenetics, 1 point for having 1-10% peripheral blasts, and 1 point for AML arising from MDS or MPD. Pts with 0 points were favorable, 1 point intermediate, 2 or more points unfavorable. Results: Of 584 AML pts identified, 361 were treated with remission induction therapy, of whom 90 had AML arising from MDS, MPD, or t-AML. Thirty-nine (43%) had antecedent MDS, 21 (23%) an MPD, and 30 (33%) had t-AML, and 47% were female. Pts with AML arising from MDS were older at AML diagnosis (median of 67 years) vs. from MPD (61 years) and t-AML (60 years) (p=.02) but a shorter time from antecedent diagnosis/event (7 months, vs. 47 and 37 months, respectively (p

Beschreibung: Tumor lysis syndrome (TLS) is a potentially lethal metabolic complication of chemotherapy or cytolytic antibody therapy usually seen in patients with hematologic malignancies, especially those malignancies with a high proliferative rate, large cellular burden and/or sensitivity to chemotherapy. The prevention and management of TLS includes hydration and reduction of serum uric acid (SUA) levels. Although Allopurinol (ALLO) has had longstanding use for TLS prophylaxis, its efficacy in controlling SUA is limited, especially due of its lack of action on pre-existing hyperuricemia. Rasburicase (RAS), a recombinant urate oxidase, effectively reduces SUA due to conversion of UA into allantoin, a readily excretable and soluble substance. RAS has significant activity in the initial management of TLS-associated acute hyperuricemia in pediatric populations, and is currently indicated in the US for this condition in children and adolescents. A prospective, randomized, controlled phase III study was conducted in adult pts to compare the efficacy in SUA control of RAS (0.20 mg/kg/d, IV) days 1–5, versus RAS+ALLO (RAS 0.20 mg/kg/d, IV days 1–3 plus oral ALLO 300 mg/day days 3–5) versus ALLO alone (300 mg/d) days 1–5. 280 pts (275 evaluable) with hematological malignancies at high or potential risk for TLS were enrolled. 92 pts received RAS, 92 pts received RAS+ALLO, and 91 received ALLO. Treatment arms were well balanced in terms of demographics, baseline characteristics, TLS risk, and percentage of pts with baseline hyperuricemia. The SUA response rate - defined as normalization of SUA (≤ 7.5mg/dl) at days 3–7 was 87.0% in the RAS arm, 78.3% in the RAS+ALLO arm and 65.9% in the ALLO arm. RAS was superior over ALLO (p=0.0009) in the overall study population as well as in pts at high risk TLS (89.0% vs. 62.8%, p=0.0012), and in pts with baseline hyperuricemia (89.5% vs. 52.9%, p=0.0151). The time to control SUA in hyperuricemic pts was 4.1 h in the RAS arm and 27 h in the ALLO arm. The mean SUA area under the curve (AUC) results indicated that there was an 8.4-fold increase in UA exposure in the ALLO arm compared to the RAS arm. There were no significant differences in the incidence or severity of adverse events, serious adverse events or deaths. The majority of RAS and/or ALLO-related adverse events were grade 1 and 2, and most of these events were hypersensitivity-related reactions. No cases of anaphylaxis, methemoglobinemia or hemolysis were observed with RAS treatment. In conclusion, RAS is superior to ALLO in normalization of SUA, with a faster effect, in adult pts at risk for TLS. RAS alone or followed by ALLO are two valid options for this patient population.

Beschreibung: E. coli. L-Asparaginase repeated injections induce immunization. Anti-Asparaginase antibodies can provoke clinical hypersensitivity reactions and/or silently inactivate enzyme activity. Consequently, L-Asparaginase clearance is increased, implying a lack of L-asparagine deamination. Firstly, we developed an assay able to detect the presence of neutralizing factors including anti-Asparaginase antibodies. Next we investigated in a mouse model if loading L-Asparaginase into red blood cells (RBC) may be a way to protect its activity against neutralizing factors. A rabbit was immunized injecting 0.5 mg of L-Asparaginase (167 IU) mixed with Freund’s adjuvant every 3 weeks for 4-fold. The animal was euthanized and the final serum collected. Part of this final serum was immuno-adsorbed onto protein A for IgG antibodies purification. L-Asparaginase activity was measured by monitoring the kinetics of ammonia generation from the hydrolysis of asparagine. This assay was adapted to a biochemistry automated analyzer. When mixed with undiluted serum from the immunized rabbit, L-Asparaginase activity (0.8 to 100 IU/ml) was totally inhibited for all the concentration range within 15 min at 37°C. In the other hand, up to 1/128 serial dilutions of serum totally inhibited 2 IU/ml L-Asparaginase. As a control, undiluted pre-immunization serum from the same animal did not significantly affect L-Asparaginase activity. To identify the neutralizing factors, IgG from serum were purified by protein-A. As performed with serum, successive dilutions of IgG were mixed with 1.25 IU/ml L-Asparaginase. The IgG inhibited enzyme activity at the 1/128 dilution by 97%, thus proving their neutralizing effect on L-Asparaginase. To simulate the presence of neutralizing antibodies in the patient, we injected 7.5 μg of rabbit IgG into OF1 mice. Control mice were injected with phosphate buffered saline (PBS). Twenty minutes later mice either received 80 IU/kg of native E. coli L-Asparaginase or the same dose entrapped into OF1 mouse RBC. L-Asparaginase was loaded into murine RBC by reversible hypotonic dialysis, followed by a resealing step. The RBC thus acts as a bioreactor where plasmatic asparagine enters and is cleaved by the entrapped L-Asparaginase inside the erythrocyte. L-Asparaginase activity inside the erythrocyte was quantified at 68 IU per ml of erythrocytes, and the extracellular enzyme activity was less to 9% of total enzyme activity. Mice were sacrificed 6 hours after the administration of native or encapsulated L-Asparaginase. Free L-Asparaginase was totally inactivated in plasma of anti-Asparaginase IgG pre-treated mice: 0.002 ±0.002 IU/ml vs 0.417 ±0.103 IU/ml in PBS pre-treated mice. In addition, when L-Asparaginase is loaded inside RBC the activity is maintained irrespective of the presence of antibodies (0.798 ±0.126 IU/ml with IgG vs 0.879 ±0.146 IU/ml without). Moreover asparagine was not deaminated in IgG pre-treated mice who received free L-Asparaginase (27 ±1.6 μmol/L), while below 2 μmol/L in all the other groups. In conclusion, this newly developed assay can predict in vivo L-Asparaginase inefficacy. In addition, L-Asparaginase loaded into RBC is protected against neutralizing antibodies and its efficacy is maintained.

Beschreibung: Reduction in bone mineral density (BMD), fractures, altered body composition, deteriorated motor performance and impaired passive ankle dorsiflexion are side-effects of chemotherapy during childhood acute lymphoblastic leukemia (ALL). Because only scarce information is available on the value of regular exercise to prevent these side-effects, we performed a randomized trial to investigate the effects of intervention with an exercise program. At diagnosis 51 ALL patients (median age: 5.4 years) were randomized into a group receiving intervention (including twice-daily high-intensity weight-bearing activities throughout the two-year during treatment period), or a control group receiving standard care. BMD of total body (BMDTB) and lumbar spine (BMDLS) and body composition parameters were measured using dual energy X-ray absorptiometry (DEXA). We measured motor performance with the Bayley Scales of Infant Development and the Movement-ABC, and passive ankle dorsiflexion with a goniometer. The investigators were blinded to the randomization. Repeated measurements analysis (ANOVA) was used. BMD decreased equally in the intervention and control group during treatment (delta-BMDTB: −0.75 SDS vs −0.96 SDS, p=0.65 and delta-BMDLS: −0.15 SDS vs −0.04 SDS, p=0.83), and increased equally during the year after treatment (delta-BMDTB: 0.42 SDS vs 0.35 SDS, p=0.70 and delta-BMDLS: 0.10 SDS vs 0.14 SDS, p=0.84). Body-fat increased during treatment in both groups (delta-fat: 1.04 SDS vs 1.56 SDS, p=0.25). In the intervention group a more rapid decline of body-fat was observed during the year after completion of therapy than in the control group (delta-fat: −1.08 SDS vs −0.49 SDS, p=0.01). Lean body mass (LBM) of both groups decreased equally during treatment (delta-LBM: −0.61 SDS vs −0.12 SDS, p=0.16) and increased equally the year after stop of treatment (delta-LBM: 0.29 SDS vs 0.22 SDS, p=0.66). Both groups showed similar changes in passive ankle dorsiflexion mobility (−5.2º vs −4.6º, p=0.76) and motor performance (0.37 SDS vs 0.68 SDS, p=0.44) during treatment. Adherence to the exercise program varied considerably: 11% of the patients performed exercises daily, 37% more than once a week, 16% once weekly and 36% less than once a week. The exercise program was not more beneficial in preventing reduction in BMD, motor performance and passive ankle dorsiflexion than standard care, most likely due to unsatisfactory compliance. However, the faster recovery after stop of treatment of the excess of body-fat in the intervention group than in the control group may be due to the educational effect of the intervention program.

Beschreibung: CD9 is a four transmembrane protein belonging to a tetraspanin family and regulates cell motility and adhesion. Several reports have indicated that CD9 form complexes with integrin including platelet fibrinogen receptor integrin aIIb-b III and involve in platelet function. We have previously reported that the c-myb knock down (KD) mice exhibited anemia and thrombocytopenia, and the expression level of CD9 mRNA was markdly increased in the c-myb KD mice. Reverse correlation of c-Myb expression with the CD9 gene expression was verified and agonistic antibody of CD9 stimulated megakaryocytic colony formation. These observations suggested that the CD9 expression was downregulated by c-myb. In our current study, we investigated the role of CD9 during megakaryopoiesis and platelet function by using CD9-null mice. Numbers of megakaryocytes and platelets, CFU-Meg, and ploidy were not different between wild-type and CD9-null mice. However, proplatelet formation (PPF) was significantly impaired in CD9-null megakaryocytes, and the size of proplatelets was smaller than those generated by wild-type megakaryocytes. Furthermore, after the bone marrow suppression with 5-fluorouracil (5-Fu), the recovery phase of platelet counts were delayed in the CD9-null mice. To clarify the reason of this platelet- recover delay, the number of bone marrow megakaryocyte was investigated using anti-vWF antibody staining, but the serial measurement of megakaryocyte number in CD9-null mice was not changed compared with wild-type mice. And also megakaryocyte ploidy was not changed in CD9-null mice compared with wild-type mice. Previous reports revealed that the cytoskeleton reorganization has a key role for the PPF formation, and Rac1-PAK1 signaling is important for actin restructure and aggregation prior to the cytoskeleton reorganization. The PPF formation was suppressed by Rac1 inhibitor. Although the mRNA expression levels of Rac1 in CD9-null mice were almost same as that in wild-type mice, PAK1 activation, major target of Rac1, was delayed in CD9-null mice compared with wild-type mice using thrombin-stimulating platelets. These results suggested that the delay of PAK1 activation cause the suppression of PPF formation in CD9-null mice. Our previous and current studies demonstrate that c-Myb suppresses the CD9 expression in a steady-state condition, while in the stress megakaryocytosis, CD9 is upregulated and acts to induce megakaryopoiesis and platelets production through Rac1-PAK1 signaling pathway. Elucidation of c-Myb-CD9 regulatory function seems to be important to understand the stress megakaryopoiesis.

Beschreibung: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T-cell neoplasm that develops only after long-term chronic infection with human T-cell leukemia virus-1 (HTLV-1). HTLV-1-specific Cytotoxic T lymphocytes (CTL) play an important role in suppressing proliferation of HTLV-1-infected or transformed T cells in vitro. The development of ATLL in approximately 2% of persons chronically infected with HTLV-1 may suggest a specific immunegenetic background predisposing to CTL failure in a proportion of HTLV-1 carriers. On the other hand, virus like particle is demonstrated to induce stronger humoral, T helper and cytotoxic T-cell responses without adjuvant. As free synthetic peptides or proteins are usually poor immunogens, the hepatitis B core (HBc) particle is a potential target carrier protein to induce immunity without use of an adjuvant. In this study, we examined the efficient induction of HTLV-1-specific CD8+ T-cell response by HTLV-1/HBc chimeric particles inserting HLA-A*0201-restricted HTLV-1 Tax-epitope without adjuvant (Figure 1). The immunization of HLA-A*0201 transgenic mice with the chimeric particle induced antigen-specific IFN-□ reaction by ELISPOT assay, whereas epitope peptide only induced no reaction (Figure 2). HTLV-1/HLA tetramer assay detected induction of HTLV-1-specific CD8+ T-cells in both splenic and inguinal lymph node cells by HTLV-1/HBc chimera particles. Furthermore, upon exposure of these dendritic cells (DCs) to the chimeric particles, the expression of CD86 was increased in a dose-dependent manner. These results suggest that HTLV-1/HBc chimeric particles are capable of inducing strong cellular immune responses without adjuvant via effective maturation of DCs and are potentially useful as an effective vaccine carrier for the treatment of cancers and infectious diseases by varying the epitope peptide. Figure 1. Electronmicrographs of recombinant HTLV-1 Tax/HBc chimeric particles. Figure 1. Electronmicrographs of recombinant HTLV-1 Tax/HBc chimeric particles. Figure 2. Induction of cellular immunity by intradermal immunization with HTLV-1/HBc chimeric particle. Mice were immunized twice with recombinant HTLV-1/HBc chimeric particle, HTLV-1 peptide and HTLV-1 peptide + HBc particle. The number of IFN-□-producing cells was measured by ELISPOT assay. IFN-□ spots show the number of peptide-loaded target cells to peptide-unloaded target cells. *P

Beschreibung: Cell membrane protein CS1 is highly expressed by tumor cells from the majority of multiple myeloma (MM) patients (〉95%) regardless of cytogenetic abnormalities and response to current treatments. Furthermore, CS1 is detected in MM patient sera and correlates with active MM. However, its role in MM pathophysiology is undefined. In the present study, we first generated CS1 null OPM2 MM cells using lentiviral CS1 short interfering RNA. Specific CS1 knockdown was confirmed by depletion of CS1 mRNA and membrane protein, whereas CS1 was expressed in parental OPM2 and OPM2 cells infected with control lentiviral vector (cntOPM2). Immunoblotting of phopho-site of multiple kinase screen analysis showed decreased phosphorylation of ERK1/2, AKT, and STAT3 in CS1null OPM2 cells vs. cntOPM2 cells. Serum deprivation markedly blocked survival at earlier time points in CS1null OPM2 cells vs. cntOPM2 cells. Earlier apoptosis in CS1null OPM2 cells correlated with earlier activation of caspases, PARP cleavage, and increased proapoptotic proteins BNIP3, BIK. CS1 knockdown further delayed development of OPM2 tumor and prolonged survival in mice. CS1null OPM2 cells failed to grow tumors in the majority of mice (n=8) at 5 weeks after cell inoculation, whereas cntOPM2 cells formed tumors within 1.5 weeks in all animals (n=8). Interestingly, CS1 was expressed in tumors that developed late in mice injected with CS1null OPM2 cells. Concomitantly, we overexpressed CS1 in CS1-low expressing U266 cells by transfecting an expressing plasmid pflagCS1 or control vector. Enforced CS1 expression enhanced U266 cell growth and survival. In contrast to the majority of U266 cells (〉95%) that grow in suspension in standard tissue culture flasks, all U266CS1 cells exhibited adherent growth and homotypic adhesion. Importantly, overexpressed CS1 increased adhesion of U266 and MM1S cells to BMSCs. Furthermore, U266CS1 cells formed more and larger colonies in methylcellulose than U266 cells. Interestingly, tumors that developed in mice injected with U266 cells expressed significantly higher levels of CS1 than injected U266 cells; moreover, exercised tumors grew in an adherent manner in vitro. Overlapping differentially expressed genes in U266CS1 vs. U266 and CS1null OPM2 vs. cntOPM2 was next analyzed by gene expression profiling. Importantly, c-maf pathway was significantly upregulated in U266CS1 vs. U266 cells and downregulated in CS1null OPM2 vs. cntOPM2 cells, as evidenced by differentially expressed c-maf and its target genes, i.e., cyclin D2, integrin αE/β7 at both mRNA and protein levels. Myeloma cell adhesion-induced VEGF secretion by BMSCs was greater with U266CS1 than U266 cells. Finally, immunoblotting showed upregulation of c-maf and cyclin D2 in U266 tumors overexpressing CS1. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

Beschreibung: B cell non-Hodgkin’s lymphoma (B-NHL) consists of different pathological entities that are frequently characterized by distinct genetic alterations. However, the knowledge on these genetic lesions in B-NHL is still limited. In order to obtain a more comprehensive view of genetic lesions in B-NHL, we performed genome-wide analysis of copy number (CN) alterations as well as allelic imbalances using Affymetrix SNP arrays in 190 B-NHL cases, including 64 samples of diffuse large B-cell lymphoma (DLBCL), 62 of follicular lymphoma (FL), 64 of mucosa-associated lymphoid tissue lymphoma (MALT-L). SNP array data were analyzed with CNAG/AsCNAR software, which enabled sensitive detection of CN alterations in allele-specific manner, and thus allelic imbalances, without depending on availability of paired normal controls. Most frequent numerical abnormalities in B-NHL were gains of chromosomes 3 and 18, although gains of chromosome 3 were less prominent in FL. Chromosomal deletions that lead to loss of heterozygosity (LOH) were commonly found in 1p, 6q and 10q. However, the more chracteristic feature of B-NHL genomes was high frequency of CN netural LOH or uniparental disomy (UPD), which was found in 35 cases of DLBCL (55%), 32 cases of FL (52%) and 18 cases of MALT-L (28%). It is widely distributed in the genome, but more frequently found in 1p, 1q, 6p, 6q and 12q. High-grade amplifications and homozygous deletions frequently provide a clue to identify relevant gene targets. In our series, 12 loci of high-grade amplifications and 14 loci of homozygous deletions were identified, and helped to specify the candidate genes. These regions included, FCGR2B amplified in 5 cases of DLBCL, RERE amplified in 2 cases of FL and CDKN2A/CDKN2B deleted in 9 cases of DLBCL. The most notable finding in the current study was, however, the identification of common genomic alterations in genes that regulate activation of NFkB pathway in more than 50% of B-NHL cases. Eight lymphoma cases harbored high-grade amplification of cREL gene, and gain including cREL was detected in 28 samples (14.7%). Fourteen cases had gains or amplification of TRAF6, and another 16 cases had deletion at 10q including PTEN. These abnormalities were supposed to cause dysregulation of NFkB. Aberrant NFkB activity has long been implicated in the pathogenesis of B-NHL, and our study confirmed that dysregulation of NFkB pathway was main mechanism of lymphomagenesis, providing further rationale that he treatment against malignant lymphoma with inhibitor of NF kappa B pathway.

Beschreibung: Identifying mechanisms of tumor-induced immune suppression will aid the development of effective immunotherapeutic strategies. In the present study we examined the molecular basis for impaired T cell responses in follicular lymphoma (FL) and demonstrate impaired T cell immunological synapse formation. Confocal microscopy was used to visualize F-actin polymerization at the immune synapse between tumor-infiltrating CD4 and CD8 T cells and autologous FL tumor cells with and without superantigen as antigen-presenting cells (APCs). We identified a significant reduction in formation of the T cell immune synapse in both CD4 (p

Beschreibung: Introduction. Previous studies clearly demonstrated the spontaneous induction and accumulation of functionally competent myeloma antigen–specific memory CD8 T cell responses in the bone marrow of a large proportion of myeloma patients. However, other studies convincingly demonstrated that CD8 T cells from myeloma patients were incabable of lysing autologous myeloma cells. An explanation for this apparent discrepancy is still lacking. CEACAMs are induced on T cells during TCR-specific activation and mediate a rapid blocking of T cell effector functions upon homophilic binding with CEACAMs expressed on target cells. We here addressed the question if myeloma cells might escape recognition by autologous, myeloma-specific CD8 T cells through CEACAM expression. Methods. Presence of myeloma-specific CD8 T cells was analyzed by IFN-y Elispot assays using separated, bone marrow derived CD8 T cells and myeloma-associated antigen-pulsed autologous DCs or autolgous myeloma cells as antigen presenting cells. Expression of CEACAMs 1–21 was analyzed by differential gene expression profiling of sorted CD138+ myeloma cells from bone marrow of 140 myeloma patients and of respective plasma cells from 14 healthy donors. In addition, the expression of CEACAMs 1, 5, 6 and 8 was analyzed by flowcytometry on the myeloma cell line MM8226 and on CD138+ myeloma cells from altogether 7 myeloma patients. A role of CEACAM on T cell recognition of autologous myeloma cells was analyzed by coculture of CD8 T cells and sorted, autologous myeloma cells in the presence or absence of blocking antibodies against CEACAMs by IFN-γ Elispot assay. Results. We identified the presence of myeloma-specifcic CD8 T cells in app. 40% of tested myeloma patients (N=20). However, in none of the tested cases T cells were able to directly recognize autologous myeloma cells. Over expression of CEACAM mRNA was found for CEACAM1, 6 and 8 in myeloma cells of up to 20% of patients, but not in plasma cells of healthy donors. Flowcytometric analysis revealed the protein expression of CEACAMs 1 and 6 on 25–50 % of the myeloma cells of all 7 tested patients and on the myeloma cell line MM8226. Blocking of CEACAMs on sorted myeloma cells before their coculture with autologous CD8 T cells resulted in significant T cell responses to myeloma cells in all tested patients (N=6), while in none of these cases the T cells were able to respond to unblocked myeloma cells. Conclusions. We here demonstrate for the first time the expression of CEACAMs 1 and 6 on freshly isolated myeloma cells. Blocking of these CEACAMs resulted in a spontaneous CD8 T cell response against cocultured autologous myeloma cells which was undetectable in case of unblocked CEACAM expression despite the presence of myeloma-reactive memory T cells. We suggest that CEACAMs on myeloma cells inhibit the re-activation of tumour antigen specific CD8 T cells upon interaction with myeloma cells and may contribute to the well characterized immune resistance of multiple myeloma.

Beschreibung: Somatic hypermutation (SHM) is a natural process that introduces point mutations into immunoglobulin (Ig) genes during antibody affinity maturation. In addition to this fundamental role in immune diversification, aberrant targeting of SHM contributes to translocations and point mutations of proto-oncogenes associated with B cell malignancy. During the first phase of SHM, the enzyme activation induced cytidine deaminase (AID) converts cytosine (C) to uracil (U) to result in a U-G mismatch. Spontaneous U-G mismatches are normally corrected by high fidelity DNA repair pathways. However, during the second phase of SHM, U-G mismatches are processed by low fidelity base excision and mismatch repair pathways to yield mutations. These second phase pathways are initiated by recognition of the uracil by uracil DNA glycosylase (UNG) and MSH2/MSH6. As a DNA mutator, AID poses a direct threat to genomic integrity but the mechanisms responsible for guiding AID to its genetic target in the first phase and determining whether a mutation will be repaired in a high fidelity or low fidelity manner during the second phase are not understood. Numerous cellular processes are regulated by small inhibitory RNA (siRNA) and microRNA (miRNA) molecules through a mechanism known as RNA interference (RNAi). To test the hypothesis that small RNA molecules are involved in SHM, we introduced two natural inhibitors of RNAi into the DT40 B cell line. DT40 cells express high levels of AID and mutate their Ig loci with high frequency. The protein 3′hExo is a siRNase that has been found to be a general inhibitor of RNAi. VA1 is a non-coding adenoviral RNA that disrupts RNAi by inhibiting nuclear export of pre-miRNA and by direct inhibition of Dicer. Following retroviral infection of 3′hExo or VA1 and empty vector controls into DT40 cells, 24 clones from each group were expanded from single cells over 28 days. DT40 cells are IgM− due to a mutation in the Ig heavy chain gene. When this mutation is corrected by AID activity, they become IgM+, allowing mutation frequency to be followed by flow cytometric analysis of IgM reversion. Compared with mock infected cells and cells infected with empty vector, cells that over-expressed 3′hExo or VA1 demonstrated a 3 and 2-fold reduction in IgM reversion, respectively. Similarly, sequencing of the Ig light chain gene from a minimum of three clones from each group revealed mutation frequencies of 6.2×10−4 mutations/nucleotide (nt) in mock infected cells, 6.8×10−4 mutations/nt in empty vector controls, 1.8×10−4 mutations/nt in cells over-expressing 3′hExo, and 2.7×10−4 mutations/nt in cells over-expressing VA1. No difference in the type of mutations or nucleotide bias was observed. Comparable reductions in RNAi activity with over-expression of 3′hExo or VA1 in DT40 cells were observed in siRNA reporter assays. Thus, general inhibition of RNAi in DT40 cells caused a 2 to 3-fold reduction in AID-mediated mutation events (P

Beschreibung: In multiple myeloma (MM), the proteasome inhibitor bortezomib induces mesenchymal stem cells (MSC) toward osteoblast differentiation. However, it is full unclear about the mechanism(s) underlying bortezomib in this process. Wnt/beta-catenin pathway plays a pivotal role in osteoblast differenciation and bone development. We have demonstrated that inhibition of Wnt/beta-catenin signaling by MM-derived Dkk1 suppresses osteoblast progenitor cell differentiation into osteoblasts (Qiang et al, Bone 2008) and deregulate RANKL and OPG expression in osteoblast cells (Qiang et al Blood 2008a). Increase Wnt signaling by overexpression of Wnt3a in myeloma cells diminished MM-trigged bone lesion in mouse model (Qiang et al Blood 2008b). In the present study we revealed that bortezomib promotes MSC differentiation into osteoblast cells via Wnt-independent activation of beta-catenin/TCF signaling. E-cadherin pull-down assay and subsequently immunoblotting analysis demonstrated that bortezomib induced increases in both free and active forms of beta-catenin protein in cytoplasm and nuclear in bell-shaped dose- and time-dependent manner in mouse and human osteoblast progenitor cell lines including C2C12, C3H10T1/2, Saos-2 and MG63. Similar results were illustrated in primary human 2 cases of normal MSC and MSC from 8 cases of MM pateints. Bortezomib induced increase in ubiquitinated beta-catenin was evidenced by obvious seen slow migration bands of beta-catenin protein in SDS-PAGE gel analysis indicating that bortezomib increased beta-catenin protein by modification of proteasome-mediated degradation of beta-catenin. Increase in cytoplasm and nuclear beta-catenin protein response to bortezomib treatment in the osteoblast cell lines and 4 cases MM derived MSC was further confirmed by immunofluorescent analysis. RT-PCT analysis of TCF family revealed that abundant TCF1 and TCF4 mRNA were expressed in all tested cell lines and in a primary normal MSC, and MM-derived MSC. Bortezomib treatment also resulted in TCF transcriptional activity in bell-shaped, dose-dependent pattern as determined by luciferase activity in these cells transfected with TOPflash plasmid DNAs. Maximal responses to bortezomib were seen at 12.5 nM for both C2C12 (p

Beschreibung: Patients with high-risk essential thrombocythemia (ET) and polycythemia vera (PV) are typically managed with cytoreductive agents such as recombinant interferon-alpha (IFN-α), hydroxyurea (HU), and anagrelide (AG). Despite the significant activity of IFN-α in ET and PV, this agent is frequently hindered by poor tolerance and inconvenient dosing schedules. PEG-IFN-α is formulated by covalently attaching polymers of ethylene glycol to the native IFN-α molecule, resulting in decreased renal excretion and increased serum half-life that allows for weekly administration. On this basis, we are conducting a phase II study of subcutaneous PEG-IFN-α-2a (Pegasys) for patients with ET or PV. A total of 76 patients have been enrolled and treated thus far (36 ET, 40 PV). Median age is 53 years (range, 18–77), time from diagnosis to PEG-IFN-α-2a 49 months (range, 0–355), WBC count 8.7×109/L (range, 3.7–27.8), hemoglobin 13.5 g/dL (range, 8.9–18.8), and platelet count 554×109/L (range, 140–1641). Prior therapies (median 1; range 0–6) included HU (n=44), AG (n=29), IFN-α (n=11: 5 oral and 6 sc), imatinib (n=7), and dasatinib (n=1). PEG-IFN-α-2a was the initial therapy in 13 patients that refused therapy with HU. The JAK2 V617F mutation was detected in 20 (56%) of 36 ET and in 37 (92.5%) of 40 PV patients. Nine (12%) patients had abnormal cytogenetics. Initial starting dose of PEG-IFN-α-2a was 450 mcg/week, but that was modified to the current starting dose of 90 mcg/week. Dose modifications are allowed according to response or toxicity. Patients are currently receiving 450 mcg (n=1), 270 mcg (n=3), 180 mcg (n=14), 135 mcg (n=8), 90 mcg (n=27), and 45 mcg (n=7). After a median follow-up of 23 months (range, 2–38), 63 (85%) of 74 assessable patients have responded. The median time to response was 4 weeks (range, 0.5–26). Complete response (CR) was achieved by 60 (81%) patients (for ET: platelets 50% reduction. In 5 (11%) of the latter the mutant allele became undetectable. PEG-IFN-α-2a was well tolerated in most patients. Thirty-nine episodes of grade 3–4 toxicity were reported: neutropenia (n=15), elevated transaminases (n=5), infection (n=4), fatigue (n=3), pain (n=3), cardiac (n=2), and anemia, thrombocytopenia, depression, shortness of breath, pruritus, thrombosis, and dizziness in 1 case each. Sixteen (21%) patients were taken off study after a median of 8 months (range, 2–26) on PEG-IFN-α-2a but only 7 (9%) of them due to due to therapy-related toxicities: grade 3 neutropenia, anorexia, depression, ischemic retinopathy, dyspnea, confusion, and pruritic rash. In conclusion, PEG-IFN-α-2a therapy results in remarkable clinical activity with an acceptable toxicity profile in advanced, previously treated, patients with ET or PV. Clinical responses are frequently accompanied by significant reduction of JAK2 V617F allele burden, which becomes undetectable in a proportion of them, suggesting selective targeting of the malignant clone.

Beschreibung: Adoptive cell transfer (ACT) of tumor-specific T lymphocytes is a powerful strategy for targeted therapy of cancer, but most of the research in this area has been focused on cytotoxic CD8+ T cells, which directly lyse MHC class I expressing targets. We have recently demonstrated that CD4+ TCR transgenic Th cells specific for self/tumor antigen tyrosinase-related protein 1 (TRP-1) have the ability to reject large established B16 murine melanoma in a model closely mimicking advanced human disease. Moreover, we showed that Th17-polarized cells were more effective in mediating complete tumor rejection than Th1-skewed cells that were capable of producing high quantities of interferon γ (IFN-γ). Interestingly, while Th1 and Th17 populations varied significantly in their phenotype, cytokines profiles, persistence and proliferation patterns in vivo, the Th17 anti-tumor function was critically dependent on the ability of the transferred cells to secrete IFN-γ. This suggests that the Th17 population might gradually acquire Th1-like properties in vivo, and that transcription factors regulating Th17 differentiation (ROR- γt) as well as IFN-γ production and Th1 polarization (t-bet) might be crucial for the effective rejection of the tumor. In order to emulate clinically relevant gene-therapy scenario we inserted TRP-1 TCR into open-repertoire CD4+ T cells from wild-type donors using a retroviral vector. Prior to transduction Th cells were stimulated under neutral (Th0) and polarizing Th1 and Th17 conditions. The majority of transduced cells expressed the Vβ14 chain, released appropriate polarization-defining cytokines upon specific antigenic stimulation in vitro and caused development of massive autoimmune vitiligo upon adoptive cell transfer into wild-type and Rag1−l/&minus mice. Gene-modified cells were readily detectable in Rag1−/− animals by flow-cytometry for more than 4 month after transfer. No off-target GVHD-like toxicities resulting from potential miss-pairing of endogenous and inserted TCR chains were observed. Th0 or Th1 and Th17-polarized TCR-transduced cells were all capable of treating mice bearing large (50–100mm2) B16 tumors, but complete cures with long-term survival occurred more robustly in animals treated with Th17-polarized effectors. To address the question whether plasticity of Th17-skewed effectors is important for their function upon ACT, we treated animals with TCR-transduced Th17-polarized cells derived from t-bet-deficient donors, which are not able to develop Th1-type responses, most importantly, not capable of producing IFN-γ. In contrast to WT-derived Th17 effectors used as a control, t-bet-deficient Th17 cells were able to mediate only minimal delay in tumor growth, suggesting that indeed the ability to acquire Th1-like properties is essential for the anti-tumor function of Th17-skewed lymphocytes. Currently, the clinical effectiveness of the ACT therapy might be hampered by the lack of high-avidity autologous effectors recognizing self/tumor antigens due to the central tolerance mechanisms. Here we demonstrate that the mature effector Th cells can be genetically engineered to express TCR recognizing MHC class II self/tumor antigen and those cells mediate powerful anti-cancer effect in vivo in a realistic model. While tumor-specific Th17-skewed CD4+ T cells are most effective in this setting, t-bet-mediated plasticity in lineage commitment is required for the full therapeutic effect.

Beschreibung: During inflammation, activated neutrophils go through the oxidative burst, releasing various oxidants, including superoxide radical, hydrogen peroxide, and hypochlorous acid (HOCl). Activated neutrophils also release myeloperoxidase (MPO), which generates HOCl from hydrogen peroxide and chloride ions. HOCl preferentially oxidizes cysteine and methionine residues to cysteine sulfenic acid and methionine sulfoxide, respectively, at rates ~100 times faster than it oxidizes tyrosine, another commonly oxidized amino acid. HOCl can also oxidize tyrosine to chlorotyrosine. Of great interest in this regard is the fact that the ADAMTS13 cleavage site in VWF, the Tyr1605–Met1606 peptide bond, contains two residues that are potential targets for myeloperoxidase-mediated oxidation. Given previous studies from our laboratory that VWF cleavage by ADAMTS13 is inhibited by oxidants, we hypothesized that neutrophil oxidants might oxidize either or both of these two amino acid residues and thereby potentially inhibit ADAMTS13-mediated cleavage. We tested our hypothesis using a peptide substrate for ADAMTS13 based on the VWF A2 sequence Leu1591–Arg1668. We incubated the VWF A2 peptide either without HOCl or with 25 or 75 μM HOCl, followed by quenching the oxidant with free methionine. The peptides were then incubated with purified recombinant ADAMTS13 and the reaction sampled every 15 min for one hour. We analyzed the cleavage reaction in two ways: by electrophoretic separation on a Tricine gel and densitometric quantification of the cleavage product, and by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) to determine the location and extent of oxidative modification and quantity of the cleavage product. We found that, after exposure to 75 μM HOCl, the A2 peptide contained methionine sulfoxide at position 1606 in 99% of the molecules in the sample, whereas only 0.3% contained both chlorotyrosine at position 1605 and methionine sulfoxide at 1606. The rate of substrate cleavage by ADAMTS13 was markedly reduced with oxidation, as measured by both assays, with the rate for the peptide treated with 75 μM HOCl being only 20% of that of the non-oxidized peptide. Taken together, these findings suggest that oxidants released by activated neutrophils during inflammation have a prothrombotic effect, mediated at least in part by inhibition of VWF cleavage by ADAMTS13.

Beschreibung: Acute lymphoblastic leukemia (ALL) is the most frequent malignant disease in childhood. Advances in therapy, in particular stratification of patients to appropriate treatment according to risk factors improved long term survival attaining cure rates of up to 80 %. Despite the efforts achieved by the stratification strategies the majority of relapse patients are recruited from the low risk groups emphasizing the need for additional independent risk factors. In a recent study we transplanted primary leukemia samples obtained from pediatric patients with newly diagnosed B cell precursor ALL (BCP-ALL) into NOD/SCID mice. Time to leukemia (TTL) was analyzed for each patient sample transplanted from date of transplant to date of leukemia manifestation in the recipients. We demonstrated that patients whose leukemia cells engrafted rapidly leading to manifestation of the disease within 10 weeks (TTLshort) showed a clearly inferior relapse free survival in contrast to patient samples with prolonged in vivo growth (TTLlong). Interestingly, the same distinct difference in relapse free survival was observed in the low risk groups only. Multivariate analysis showed an almost 45- fold increased risk for relapse in TTLshort patients. In order to further characterize the biological properties of the leukemia cells in the two groups, gene expression profiles of samples with short versus long TTL in the xenograft model were analyzed using a human whole genome array (Affymetrix U133 Plus 2.0). Here, we used quantitative traits analysis (QTA) correlating gene expression values (relative expression) to the time from transplant to manifestation of leukemia in the NOD/SCID mice (TTL, in weeks). 14 different xenograft samples (TTLshort n= 7, mean TTL 8.14 weeks; TTLlong n= 7, mean TTL 19.71 weeks; T-test P = .03) isolated from leukemia bearing mice were investigated. All 14 patients included were stratified in low (standard or intermediate) risk groups. All patients were negative for TEL/AML1- fusion, BCR/ABL- fusion or MLL rearrangement. By QTA we identified 5 genes that were significantly correlated (Spearman correlation, for all 5 genes P 〈 .0001) to time from transplant to leukemia manifestation in the recipient mice (TTL). Analysis of the 14 xenograft samples using the 5 genes identified showed clustering of all but one sample in one group. The 5 genes were than explored for their power to predict relapse in an independent cohort of pediatric ALL patients. For these patients expression profiles were analyzed in leukemia samples obtained at diagnosis. All patients in this cohort were also stratified in low risk groups and negative for the presence of TEL/AML1-, BCR/ABL- or MLL- fusion transcripts. 8 of the 38 patients encountered relapse, 5 at early and 3 at late time points (〈 or 〉 24 months after diagnosis). Clustering according to the 5 genes identified by QTA lead to separation of the patients into two groups. Interestingly, all relapsed patients except one clustered in the group associated with the signature characteristic for TTLshort. Most importantly, all 5 patients with early relapse gathered in this cluster. Taken together, we used a novel approach directly correlating gene expression values to the continuous variable time to leukemia (TTL) which might be reflected more accurately by this strategy than comparing two groups. We applied this gene signature characteristic for TTLshort, thereby identifying poor overall survival in a set of independent pediatric ALL patients and found clustering of all early relapse patients in one group. This new independent risk factor might identify patients with high risk for early relapse avoiding xenografting in the mouse model.

Beschreibung: Erlotinib, an inhibitor of the epidermal growth factor receptor (EGFR), induces differentiation, cell-cycle arrest, and apoptosis of EGFR-negative myeloblasts of patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), as well as in EGFR-negative cell lines representing these diseases (P39, KG-1, and HL 60). This off-target effect can be explained by inhibitory effects on JAK2. Apoptosis induction coupled to mitochondrial membrane permeabilization occurred independently from phenotypic differentiation. In apoptosis-sensitive AML cells, erlotinib caused a rapid (within less than 1 hour) nucleocytoplasmic translocation of nucleophosmin-1 (NPM-1) and p14ARF. Apoptosis-insensitive myeloblasts failed to manifest this translocation yet became sensitive to apoptosis induction by erlotinib when NPM-1 was depleted by RNA interference. Moreover, erlotinib reduced the growth of xenografted human AML cells in vivo. Erlotinib also killed CD34+ bone marrow blasts from MDS and AML patients while sparing normal CD34+ progenitors. This ex vivo therapeutic effect was once more associated with the nucleocytoplasmic translocation of NPM-1 and p14ARF. One patient afflicted with both MDS and non–small cell lung cancer manifested hematologic improvement in response to erlotinib. In summary, we here provide novel evidence in vitro, ex vivo, and in vivo for the potential therapeutic efficacy of erlotinib in the treatment of high-risk MDS and AML.

Beschreibung: The homeobox transcription factor Cdx2 is one of the most frequent ectopically expressed proto-oncogenes in human AML and when retrovirally expressed causes AML in mice (Rawat et. al. PNAS 2004 and Blood 2008). As the leukemogenic potential of Cdx2 was dependent on its N-terminal transactivation domain, we now extended structure-function analyses by inactivating the evolutionarily conserved MAPK phosphorylation sites (S60, S99, S108, S156, S60-S99-108 (3S), and S60-S99-108-156 (4S)) in Cdx2. Peripheral blood analysis of the mice, transplanted with bone marrow cells retrovirally transduced with different Cdx2 serine mutants (n=5) after six weeks, revealed that all the Cdx2 serine mutants showed significant growth advantage over non-transduced carrier cells. However, assessment of engraftment after 16 weeks showed that mice transplanted with BM expressing the S99, the S108 mutants, the 3S and 4S failed to develop leukemic engraftment in contrast to wild type Cdx2, indicating that the S99 and S108 serine sites are critical for leukemic transformation. Furthermore, mice transplanted with BM expressing Cdx2 wild type (n=24), the S60 (n=3) or S156 mutant (n=5) developed AML after median latency of 120, 90 and 167 days post transplantation, respectively. In contrast S99, S108 and 3S mice developed AML after very long latency of 328 (5/5 mice), 341(3/5 mice) and 336 (3/5 mice) days post transplantation, respectively. Interestingly, 4S mice (n=5), did not develop any disease up to an observation time of 400 days, indicating that the transforming potential of Cdx2 was dependent on multiple N-terminal serines. As it was shown that the transcriptional activity of Cdx2 is dependent on the phosphorylation status of N-terminal serines in non-hematopoietic tissue we tested the phosphorylation status in hematopoietic cells: murine cells retrovirally transduced with Cdx2 and human AML cell lines positive for CDX2 expression showed a phosphorylated form of CDX2 and an activated Erk1/2 pathway, in contrast to AML cell lines negative for CDX2 expression. Based on this we tested whether inhibition of the MAPK pathway would impair the transforming potential of Cdx2. When Cdx2 transduced BM cells were incubated with MEK1/2 inhibitors (PD98059, U0126) for 14 days in liquid culture, viability of the cells was reduced by 78% (n=3, p

Beschreibung: Roughly three-quarter of the genes associated with human diseases have fly counterparts. This high degree of conservation, combined to a wide range of genetic tools, makes Drosophila an attractive model to study basic mechanisms lying at the heart of various human disorders. Several oncogenes mediate their effects by interfering with specific cell machinery components common to all eukaryotes. The systematic identification of cell components influencing the activity of oncogenes should therefore accelerate the characterization of those oncogenes. Toward this goal, we take advantage of Drosophila molecular genetics to identify conserved genes that functionally interact with oncogenes. Our effort currently focuses on the t(7;11)(p15;p15) translocation associated with acute myeloid leukemia (AML) and which fuses the N-terminal part of Nucleoporin 98 (NUP98) to the C-terminal part of the transcription factor HOXA9. As homologues of NUP98 and HOXA9 are present in flies, we hypothesized that expression of NUP98-HOXA9 during development will affect some of the same protein networks that are perturbed in human hematopoietic cells. We successfully conducted several modifier screens in the past by exploiting dosage-sensitive phenotypes specifically induced in the eyes. To that end, we expressed NUP98-HOXA9 during eye development, which interestingly phenocopied Homothorax (HTH) overexpression in its ability to block eye development and promoted head cuticle formation. HTH is the homologue of MEIS1; a DNA-binding co-factor for HOXA9 that functions with a third partner, PBX, and which together form a ternary complex that regulates gene expression. Importantly, we found that the NUP98-HOXA9 eye phenotype was suppressed by mutations in the hth and exd (Drosophila pbx) genes, thus lending support to the specificity of the phenotype. In agreement with this, a structure/function analysis of NUP98-HOXA9 conducted in the fly eye narrowed down the same functional domains/motifs as those that had been identified using mouse models, namely, the HOXA9 homeodomain, the HOXA9 ANW motif (a PBX-interaction site) and the NUP98 portion. Remarkably, we also found that NUP98-HOXA9 and HTH/MEIS synergistically induced cell proliferation when coexpressed in the developing eye. As a result, large tissue overgrowths were produced. The cooperation observed in this experimental setting is reminiscent of the ability of MEIS1 to accelerate AML onset when co-expressed with NUP98-HOXA9 in mouse models. Moreover, we found that the collaboration strictly depends on endogenous EXD/PBX as its depletion by RNAi completely prevents overgrowth formation. Together, these findings provide compelling evidence that the NUP98-HOXA9 fly model recapitulates several of the key functional features that had been established in mammals for this oncogene and thus should prove useful to further delineate the immediate events disturbed by NUP98-HOXA9 expression. Based on these premises, we conducted a genetic screen to isolate dominant modifiers of the NUP98-HOXA9 eye phenotype. Approximately 100,000 fly progeny have been screened, which led to the isolation of a few hundred mutations acting either as suppressors or enhancers. Several complementation groups have now been uncovered and their molecular identification is currently underway. Validation of relevant genes in mouse leukemia models will be conducted to confirm their significance with respect to HOX-dependent leukemia. The NUP98-HOXA9 fly model as well as the early findings of the screen will be presented.

Beschreibung: Background: B-cell non-Hodgkin lymphomas (NHL) are common lymphoid malignancies in which malignant B-cells arrested at various stages of differentiation proliferate within lymph nodes and occasionally other tissues. The microenvironment that supports the growth and survival of NHL B cells consists of a complex network of immune cells and cytokines and its specific composition has been shown to have significant clinical implications. The intratumoral immune cells include effector and regulatory T (Treg) lymphocytes that are more than simple residual elements from the normal lymph node structure. We previously explored whether the extent of T cell infiltration played a role in the clinical outcome of patients with B-cell NHL and found that an increased percentage of CD4+ T cells in the diagnostic biopsy of lymphoma patients significantly correlated with an improved 5-year overall survival. We have also shown that intratumoral Treg cells significantly suppress the anti-tumor response. Based on the fact that intratumoral T-cells are important in B-cell NHL, we evaluated whether genetic variation in genes that regulate the T-cell response may be associated with lymphoma risk. Methods: We genotyped 257 single nucleotide polymorphisms (SNPs) from 50 candidate genes related to T-cell differentiation and function in a clinic-based study of 441 Caucasian NHL cases and 475 frequency matched Caucasian controls seen at the Mayo Clinic from 2002–2005. Tagging and nsSNPs were selected from HapMap. The most prevalent homozygous genotype was used as the reference group and each SNP was modeled individually as having a log-additive effect, expressed as ordinal odds ratios (OR) per variant allele and 95% confidence intervals, in an age- and sex-adjusted logistic regression analysis. We also evaluated the consistency of the findings for the most common types of B-cell NHL - diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and CLL/SLL. For gene-level analyses, we used principal components (PC) analysis using all SNPs from a gene; those PCs that explained 〉 90% of the variability were then modeled in a logistic regression analysis and significance was determined using a likelihood ratio test. We also evaluated the association of haplotypes from each gene with risk of NHL using the score test implemented from HAPLO.SCORE. Results. The mean age at diagnosis was 60.1 years for cases and 58% were male; among controls, the mean age at enrollment was 61.7 years and 55% were men. In the PC gene analyses, PRF1 (perforin gene - involved in cytotoxicity; p=0.004), CD276 (B7-H3 gene - involved in co-stimulation; p=0.01), TBX21 (T-bet gene – regulates Th1 cell development; p=0.02), and IL6 (p=0.02) were significant at p

Beschreibung: Background: Expression of Human herpesvirus 8 (HHV-8) K1 causes hyperplasia of lymph nodes and lymphomas in mice. The exact mechanism of how K1 causes hyperplasia and lymphomas in K1 expressing mice is not known. The cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM) of K1 was shown previously to be involved in activation of Nuclear Factor kappa B (NF-k-B). Moreover, we had shown recently that K1 suppresses Fas-mediated apoptosis through its extracellular immunoglobulin-like domain and that K1-transfected mice survived a lethal dose of agonistic anti-Fas antibody (Jo2). We thus hypothesized that development of hyperplasia and lymphomas in K1-expressing mice is driven by alterations of Fas signaling. Results: At 18 months, 10 K1 transgenic mice, 90% developed lymphoid hyperplasia (〉3mm) and 60% developed lymphomas, while all (26) control mice remained hyperplasia and lymphoma free. In the extreme cases, K1 mice developed liver or mesenteric tumors (4 and 4 of 10 mice, respectively). Spleens of 78% of K1 mice were enlarged at 18 months and were on average 3.5 times heavier than spleens of non-expressing control mice (332 ± 200 mg vs. 94 ± 26 mg, P 〈 0.03). The H and E staining of spleen sections showed expansion to the periarteriolar lymphocyte sheath with disruption of normal follicular architecture. Staining of spleen sections with anti-kappa and anti-lambda light chain antibodies revealed presence of monoclonal foci in 3 out of 3 K1 mice (average 6 foci per single section of spleen), but none in the 4 control mice. Moreover, K1 protein was expressed in about 10% of splenic cells as judged from staining with anti-K1 antibody 2H5. To test the hypothesis that expression of K1 protein in spleens makes them resistant to Fas-mediated apoptosis, splenic cells of 6 month old K1 mice (n=3) and matched controls (n=3) were isolated and incubated with 50 ng/mL of agonistic anti-Fas antibody Jo2. At 12 hours of treatment, only 4 ± 1% of splenocytes from K1 mice versus 17 ± 2% of control splenocytes were undergoing apoptosis (P

Beschreibung: Introduction: Certain autoimmune and infectious conditions are associated with increased risks of subtypes of non-Hodgkin lymphomas (NHL). A few prior studies suggest that chronic immune stimulation may particularly elevate risk for the NHL subtype lymphoplasmacytic lymphoma (LPL)/Waldenström’s macroglobulinemia (WM). To improve our understanding on the role of immune-related and inflammatory conditions in LPL/WM pathogenesis, we conducted a large population-based study including close to 2,500 LPL/WM patients diagnosed in Sweden and almost 10,000 matched controls. Methods: Using both the central Cancer registry and local hospital-based registries, we identified all LPL/WM patients diagnosed in Swedish hospitals 1958–2005. From the Swedish Population Registry we identified four matched controls per LPL/WM patient. Through data linkage with the central Inpatient registry, we gathered information on hospital inpatient discharges that listed autoimmune-, infectious-, and other inflammatory/allergic diseases present at least 1 year prior to LPL/WM. Using Poisson regression, we calculated rate ratios (RR) and 95% confidence intervals (CI) adjusted for categorical year of birth, date of diagnosis, gender, and county. Results: A total of 2,470 LPL/WM patients (647 LPL and 1,823 WM), and 9,698 population-based matched controls were included in the study. We found an increased risk of developing LPL/WM among individuals with a prior history of systemic sclerosis (RR=4.7; 1.4–15.3), Sjögren’s syndrome (RR=12.1; 3.3–45.0), autoimmune hemolytic anemia (AIHA) (RR=24.2; 5.4–108.2), polymyalgia rheumatica (RR=2.9; 1.6–5.2), and temporalis arteritis (RR=8.3; 2.1–33.1). We also found excess risk of LPL/WM among persons with a history of pneumonia (RR=1.4; 1.1–1.7), septicaemia (RR=2.4; 1.2–4.3), pyelonephritis (RR=1.7; 1.1–2.5), sinusitis (RR=2.7; 1.4–4.9), herpes zoster (RR=3.4; 2.0–5.6), and influenza (RR=2.9; 1.7–5.0). Importantly, when we assessed the associations by latency (time between immune-related or inflammatory conditions and LPL/WM), for most autoimmune- and infectious diseases the excess LPL/WM risk remained significant at 〉5 years latency. We found no significant increased risk for LPL/WM among individuals with prior chronic inflammatory or allergic conditions. Conclusions: In the largest investigation of risk factors for LPL/WM to date, we found a personal history of certain autoimmune and infectious diseases to be associated with excess LPL/WM risk. Immune-related conditions might act as potential LPL/WM triggers or they could represent premalignant immune disruptions preceding LPL/WM. Our results provide novel insights into the as yet unclear pathogenesis of LPL/WM.

Beschreibung: Background: Hypogammaglobulinemia of the “uninvolved” immunoglobulins is commonly observed in Waldenstrom’s macroglobulinemia (WM), and has often been attributed to disease-related suppression. However, there is a paucity of information related to the pathogenesis of hypogammaglobulinemia in these disorders. Methods: We evaluated the incidence of IgA and IgG hypogammaglobulinemia in 207 patients with WM, and addressed the impact of therapy and response on IgA and IgG levels for 93 of these patients who required subsequent treatment. We also performed extensive sequence analysis of the promoter, all exonic, and flanking intronic regions from peripheral blood of 19 untreated WM patients who demonstrated IgA and/or IgG hypogammaglobulinemia for 8 genes often observed in common variable immunodeficiency disorders (CVID) and B cell deficiency i.e. AICDA, BTK, CD40, CD154, NEMO(IkBkG), TACI, SH2D1A, UNG. Results: At baseline, 120/207 (58.0%), 131/207 (63.3%), and 102/196 (49.3%) patients had abnormally low levels of serum IgG (

Beschreibung: The p53 gene, metaphorically named “the Guardian of the Genome“ by David Lane in 1992, is one of the most important decision makers inside the cell. By interacting with an array of downstream genes, p53 can regulate cell fate, either by precipitating events leading to cell death by apoptosis, or by acting as a regulatory factor during G1/S phase allowing the cell to repair minor damage to DNA and proceed to the next stage of cell division. Playing such a pivotal role within the cell, p53 itself is subjected to a tight and orchestrated control, creating a network of positive and negative regulations via a number of interacting proteins (MDM-2, Wip-1, Cyclin G, p14ARF). Yet another level of p53 regulation is represented by post-translational modifications, modulating its transcriptional/transactivational ability. These modifications include phosphorylations on serines 15, 18 and 20 or acetylations at C-terminal lysines of p53. Recently, it has been found that the regulation of p53 is also substantially controlled at the transcriptional level. They identified nine different splicing variants of p53 with distinct biological characteristics, resulting from combinations of an alternative splicing of intron 2, 9 and/or aberrant transcription, starting at so-far unrecognized cryptic promoter in intron 4. Herein we present evidence of a novel splicing variant of p53 gene, termed delta ex6 that is differentially expressed in patients with chronic lymphocytic leukemia (CLL) as compared to healthy donors. The delta ex6 variant was identified in 109 out of 127 (86%) CLL patients, while in healthy individuals it was not detected. Delta ex6 variant is devoid of transactivational activity as determined in vitro by FASAY (Functional Analysis of Separated Alleles in Yeast). To test the biological properties of the delta ex6 variant we have cloned its whole coding sequence and transfected the p53-double-knock-out model cell line H1299 to produce stable integrants. Stable H1299 cell lines expressing the delta ex6 variant were distinguished by a remarkable loss of intercellular contacts and semi-suspension growth properties, in contrast to the strictly adherent growth of the parental cells and mock-transfected cells. Four stable delta ex6 producing H1299 cell lines, as well as control cell lines in doublets (parental H1299 harboring the cloning vector, and H1299 stably transfected with wild type p53) were subjected to the Affymetrix GeneChip Human Exon 1.0 ST array expression analysis. The microarray data corroborated the accented and proliferative phenotype as observed in vitro in the tissue culture: overexpression of a number of cyclins (A1, G1, G2, F, I, B2, A2, T2), matrix metalloproteinases, hyaluronidases and caspase inhibitors; and downregulation of adhesion molecules and molecules of the intercellular matrix. Our data on the presence of the delta ex6 p53 variant in CLL patients supports the recent evidence on dysregulation of p53 splicing pattern in malignancies. Moreover, as assessed in vitro, overexpression of the delta ex6 variant leads to an accented and proliferative phenotype, a finding further supporting the biological role of the novel delta ex6 p53 variant in vivo.

Beschreibung: Subcutanous panniculitis–like T cell lymphoma (STCL) is a rare clinical entity which simulates panniculitis and can present with an aggressive clinical course. STCL is divided two major subgroups (αβ-STCL and γδ-STCL), according the T cell receptor expression on the malignant T cells. γδ-STCL is often associated with a more aggressive course and poor prognosis with an 11% 5-yr survival in a retrospective study of 20 cases, only 6 of which were confirmed by TCRδ -1 staining.1 We report our results from 10 patients with STCL, 2 BetaF1+ (αβ-STCL), 8 γδ-STCL. The median age at presentation was 43 (25–63); 7 of 10 were female. Immunohistochemical studies and TCR gene rearrangements (TCRR) were performed on all patients. Cytotoxic T-cell markers were expressed in all pts (TIA-1 in 8 of 10 and Granzyme B in 5). Six were CD8+ and 3 were CD3+CD4−CD8−. CD56 expression was detected in 3. All demonstrated clonal TCRR. All pts presented with skin nodules or ulcerations, and 3 had visceral involvement (blood/bone marrow in 2, liver in 1). Three patients (2αβ-STCL and γδ-STCL) were treated initially with denileukin diftitox; one each with αβ- and γδ- disease had PR on therapy and have been maintained without PD. Seven patients were treated with cytotoxic chemotherapy regimens. Four of 7 achieved a remission after EPOCH (2), denileukin diftitox-CHOP (1) or pentostatin/cyclophosphamide followed by alemtuzumab (1). Four pts (1 with refractory ab-STCL, 2 with refractory gd-STCL, and 1 with γδ-STCL in first CR after denileukin diftitox-CHOP) underwent allogeneic hematopoietic stem cell (HSCT) from matched-related donors. Two patients are alive 6 and 13 months after HSCT with no evidence of disease; 1 patient died in CR from infectious complications of HSCT, and 1 relapsed 6 mo after HSCT and died from PD. At a median follow up of 3 yrs from diagnosis, 8 pts (80%) are alive, including the 2 pts with αβ-STCL and 6 of 8 pts with γδ-STCL. In summary, while γδ-STCL is reported in retrospective studies to have a poor prognosis, we demonstrate that aggressive therapies along with the incorporation of novel T-cell directed agents such as denileukin diftitox and alemtuzumab into treatment regimens and the use of allogeneic HSCT as a potentially curative therapy are promising approaches for these patients.

Beschreibung: Background. Measurement of serum M-spike is used to determine response to therapy and treatment free survival in Waldenstrom Macroglobulinemia (WM). However, there are many limitations to the use of IgM M-spike, and new sensitive markers are needed to determine early response or progression in these patients. We have previously shown that involved serum free light chain (sFLC) accurately diagnosed patients with WM, and correlated with markers of poor prognosis, specifically beta- 2 microglobulin (B2M). In this study, we sought to determine the role of levels of involved in the response to therapy in patients with WM treated on clinical trials, and compare it to the response observed using the traditional M-spike measurement. Methods. We prospectively studied involved sFLC in 61 WM patients enrolled on 2 clinical trials, at diagnosis (N=8) and with relapse/refractory disease (N=53). Patients were treated on one of two clinical trials: either perifosine (N=30; given 150mg oral daily) or combination of bortezomib and rituximab (N=30; given IV bortezomib 1.6mg/m2 at days 1, 8, 15 q 28 days × 6 cycles and rituxan 375 mg/m2 at days 1, 8, 15, 22 on cycles 1 and 4). Response was assessed after cycle 2, confirmed on 2 consecutive measurements, and included minor response (MR), partial response (PR) or complete remission (CR). Results. The baseline characteristics of the patients were as follows: the median age was 65 years (44–83), male/female ratio 2.38, serum B2M 3.0mg/L (1.00–7.30), hemoglobin 11.0g/dL (7.00–14.90), platelet count 216 ×109/mm3 (46–563), serum M-spike 2.24g/L (0.41–4.62), and involved sFLC 95.2mg/L (4.92–868). The WM International staging system (WM-ISS) showed that 28% of patients had low ISS, 31% intermediate, and 41% high ISS scores. The overall response rate for the entire cohort was 62.3%, assessed by M-spike measurement (MR=23, PR=13, nCR=2). The overall response rate using involved sFLC level was 72.1% (MR=14, PR=29, and CR=1), (p

Beschreibung: Fusion genes involving imatinib-sensitive (PDGFRA, PDGFRB) and imatinib-resistant (FGFR1 and JAK2) tyrosine kinases (TK) have been identified in a substantial proportion of patients with eosinophilia-associated myeloproliferative neoplasms (Eos-MPNs). They result in constitutive activation of the corresponding TK moiety by dimerization domains of the partner gene or loss of the autoinhibitory WW-like domain within the juxtamembrane region. We here present three new fusion genes with involvement of PDGFRB. Two male patients (51 and 42 years old) presented with chromosomal aberrations involving chromosome bands 5q31-33; t(5;17)(q33-35;q11.2) and t(5;20)(q33;p12). In patient #1, LDI-PCR (Walz et al., Haematologica2007:92,163) identified an in-frame fusion between myosin XVIIIA (MYO18A) exon 40 and PDGFRB exon 10. Activation is likely to occur through dimerization as the autoinhibitory WW-like domain of PDGFRB is fully retained in the fusion protein. In patient #2, 5′-RACE-PCR of mRNA identified an in-frame fusion between D-tyrosyl-tRNA deacylase 1 (DTD1) exon 4 and a truncated PDGFRB exon 12. DTD1 potentially lacks known dimerization motifs suggesting that the disruption of the autoinhibitory WW-like domain region solely contributes to enhanced TK activity. Male patient #3 (42 years old) had a dry tap due to marked myelofibrosis and cytogenetic analysis could only be performed after centrifugation of bone marrow biopsy cells. Four metaphases were obtained which all showed a normal karyotype. In Eos-MPN with normal, low quality or missing karyotype, we routinely perform quantitative RT-PCR for 3′-sequences of PDGFRA and PDGFRB which are retained in all known fusion genes. Overexpression of mRNA was shown in all samples with variable PDGFRA (5 different fusion genes in 50 samples) or PDGFRB (5 different fusion genes in 8 samples) fusion genes as compared to samples from HES or reactive eosinophilia (ratio PDGFRA/ABL1 0.73 vs. 0.0066 vs. 0.0064, p

Beschreibung: Introduction: Optimal management of mantle cell lymphoma (MCL) remains undefined. However, recent data support the conclusion that upfront consolidation with high dose therapy with autologous stem cell rescue (HDT/ASCR) can improve the PFS (Dreyling, et al., 2005; Geisler, et al., 2008) and possibly OS (Geisler, et al. 2008). Based on gene expression profiling data, the proliferation signature has emerged as a critical determinant of prognosis in MCL (Rosenwald, et al., 2003). Immunohistochemical assessment of proliferation as estimated by MIB-1 (Ki-67) staining has been shown to predict outcome in patients with MCL treated with chemotherapy or immunochemotherapy. With conventional chemotherapy, the reported MIB-1 staining cutoff associated with poor prognosis reportedly varies from 10–30%. Since 1994 we have treated patients (pts) with MCL with sequential chemotherapy followed by HDT/ASCR. The current analysis is a retrospective review of our treatment program focused on the impact of upfront consolidation with HDT/ASCR on the prognostic significance of proliferation as measured by MIB-1 immunohistochemistry. Methods: Seventy-nine patients underwent upfront consolidation with HDT/ASCR for MCL. Fifty-two patients had evaluation of proliferation by MIB-1 immunohistochemistry. MIB-1 expression was estimated visually and assigned a percentage. Patients were binned into quintiles of MIB-1 expression: 0–20% (n=32, 61.5%); 21–40% (n=5, 9.6%); 41–60% (n=5, 9.6%); 61–80% (n=5, 9.6%); and 81–100% (n=5, 9.6%). Outcomes were analyzed by the method of Kaplan-Meier and comparisons were by log-rank. Results: The EFS and OS at 5 years was 65.1% and 61.4% respectively for the subset of patients with available MIB-1 staining (n=52); the outcomes for the entire group (n=79) did not differ from the subset. Outcomes (PFS, OS) were evaluated by MIB-1 quintile with successive cutoff values of 20%, 40%, 60% and 80%. MIB-1 cutoff values of 40% or less were not predictive of outcome. However, when the cutoff was 60% or 80%, both the PFS and OS were significantly worse for the high proliferation group. Nineteen percent of patients had a proliferation fraction of greater than 60%. For patients with MIB-1 staining of 〉60% the PFS was 4.2 years and was not reached for the group with staining ≤60% (p=0.004). Similarly, overall survival was significantly different, (p=0.031). Conclusions: These findings suggest that upfront consolidation with HDT/ASCR can partially overcome the adverse impact of proliferation on outcome and the adverse outcomes are seen in a subgroup of approximately 20% of patients with MIB-1 staining of greater than 60%.

Beschreibung: Janus kinases (JAK) comprise a small family of cytoplasmic protein tyrosine kinases, which play an important role in the initiation of cytokine-triggered signaling events via signal transducer and activator of transcription (STAT) proteins. The recent reports of an activating somatic mutation in codon 617 of the JAK2 gene (JAK2V617F mutation) in patients with myeloproliferative disorders (MPDs), has opened new avenues for the development of targeted therapies for these malignancies and clinical trials with JAK2 inhibitors are underway. We report here the activity of Atiprimod (N,N-diethyl-8-dipropyl-2-azaspiro[4,5]decane-2-propanamine), a novel compound with anti-inflammatory properties, in retrovirus-transduced JAK2V617F mutant-expressing murine FDCP-EpoR cells, set-2 cells, and blood cells from patients with polycythemia vera (PV). We compared the growth inhibitory effect of Atiprimod against two mouse FDCP cell lines transfected with erythropoietin receptor (Epo-R), and either wild-type JAK2WT or mutant JAK2V617F, and human megakaryoblastic leukemia cells with mutated JAK2V617F (set-2 cells). The growth inhibitory effect was assessed using 3-days MTS assay. Atiprimod was more potent against FDCP cells carrying mutant JAK2V617F cells (IC50 0.42 μM) and set-2 cells (IC50 0.53 μM) than FDCP wildtype JAK2WT cells (IC50 0.69 μM). Atiprimod inhibited the phosphorylation of JAK2 and downstream STAT3, STAT5, and AKT proteins in a dose- and time-dependent manner. It induced apoptosis, as evidenced by increase in mitochondrial membrane potential, caspase3 activity, and cleavage of PARP protein. The anti-proliferative effect on expanded PV patient progenitor’s cells was paralleled by a decrease in JAK2V617F mutant allele frequency in BFU-E or CFU-GM clones in clonogenic assay. However, co-culturing of JAK2V617F mutant cells with three different bone marrow stromal cell lines (Hs5, ABM-MSC, NK-Tert) either directly (cell on cell) or indirectly (separated by 0.4 μm micropore membranes) for 48 hours resulted in a significant protection of mutant cells from the effect of Atiprimod. Co-culturing of bone marrow stromal cells prevented Atiprimod (0.4 and 0.8 μM) induced apoptosis, and reversed the inhibition of phosphorylation of STAT proteins. Our results suggest that cytokines secreted by stromal cells might play an important role in protecting the hematopoietic cells from a JAK2 inhibitor. Further dissection of the nature of interactions between JAK2V617F mutant cells and marrow stromal cells may lead to new therapeutic avenues for patients with MPD.

Beschreibung: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous category of lymphoid tumors that comprises different clinical forms not fully recognized in the WHO classification. In this regard, extranodal (EN) DLBCLs have particular clinicobiological features and outcome, sometimes related to the specific site where the lymphoma arises. Nowadays, rituximab plus chemotherapy (CT) is the gold-standard in the treatment of DLBCL. However, the superiority of rituximab-CT (R-CT) over CT alone has not been addressed for all the clinical subsets of the disease and, in fact, the clinical role of the new therapies might be different for primary nodal or EN DLBCLs. The aim of this study was to assess the impact of rituximab in patients suffering from nodal or EN DLBCL. Two-hundred and thirty non-immunocompromised patients (112M/118F; median age, 61 years) diagnosed with CD20+DLBCL in a single institution between 1997 and 2006 (five years before and after establishing R-CT as the standard treatment in DLBCL) and treated with adriamycin-containing regimens were the subject of the present study. The series included 148 primary nodal and 82 EN DLBCL. Patients with primary CNS lymphoma were excluded and lymphomas arising at Waldeyer ring were considered as nodal DLBCL. The main EN sites were GI (n=26), bone (n=13), soft tissue (n=13), lung/pleura (n=9), liver (n=9), and other (n=12). Main clinico-biological and evolutive variables were analyzed. One hundred nineteen patients received only CT and 111 R-CT. Eighty-seven cases with available information were assigned to germinal center B-cell-like (GCB) (41%) or non-GCB (59%) groups according to the Hans method (Blood2004;103:275–82) based on CD10, BCL6 and MUM1 expression. Main initial features, including the primary nodal or EN origin, international prognostic index (IPI), and GCB/non-GCB categories were similar for CT and R-CT groups. No correlation was observed between the GCB/non-GCB groups and the primary site of the tumor, although nodal lymphomas more frequently expressed MUM1 than EN (69% vs. 31%, respectively; p=0.01). CR rate and 5-year overall survival (OS) according to the treatment arm (CT vs. R-CT) is detailed for the whole series and for the nodal and EN groups in the table and OS curves depicted in the figure. In the whole series, variables predicting poor OS in the multivariate analysis were high-risk IPI (RR 2.5; p

Beschreibung: Burkitt lymphoma (BL) is a highly aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for nearly 3% of all newly diagnosed NHLs. Treatment of adult non-HIV-related BL with the intensive CODOX-M/IVAC regimen (modified Magrath regimen) produces complete responses (CR) in 75 to 86% of patients, with lower CR rates reported in HIV-related BL. The CD20-directed monoclonal antibody rituximab has never been reported in combination with CODOX-M/IVAC, and here we report the first series in BL patients, with or without HIV infection. A total of 24 patients were identified at our institutions who received rituximab plus CODOX-M/IVAC with curative intent. Rituximab was administered at 375mg/m2 on day 3 of cycle 1, and then on day 1 of subsequent cycles. Twenty-three patients received 4 alternating cycles of R-CODOX-M/R-IVAC for high risk disease, while 1 patient received 3 cycles of R-CODOX-M alone for low risk disease, defined as a single focus less than 10cm with a normal LDH. All patients received white cell growth factor support, and pneumocystis prophylaxis was included for all HIV+ patients. The median age was 45 years (17–67), advanced Ann Arbor stage 80%, LDH 〉 upper limit of normal 80%, extra nodal involvement 80% and ECOG PS

Beschreibung: Pralatrexate (10-propargyl-10-deazaaminopterin, PDX) is a novel antifolate with greater affinity for the reduced folate carrier (RFC-1) and foly-polyglutamyl synthase (FPGS). PDX is emerging as a promising drug for the treatment of chemotherapy resistant T-cell lymphomas and leukemias. Bortezomib (B) is a modified dipeptidyl boronic acid that induces apoptosis by inhibiting the 26S proteasome in a variety of hematologic malignancies, including multiple myeloma and non-Hodgkin’s lymphoma. Recently, NF- B has shown a prominent role in inducing resistance to apoptosis in cutaneous T-cell lymphoma (CTCL), which supports a potential therapeutic role for bortezomib in the treatment of these patients. We investigated the in vitro and/or in vivo activity of PDX and B alone or in combination in different T cell lymphoma and leukemia cell lines including CTCL (H9), T- acute lymphoblastic leukemia (T-ALL) lines resistant or sensitive to gamma- secretase inhibitors (GSI) (P12, PF 382 and CEM are GSI resistant while KOKPT-1, DND-41 and HPB-ALL are GSI sensitive lines). For all cytotoxicity assays, luminescent cell viability was performed using CellTiter-Glo™ followed by acquisition on a Biotek Synergy HT. Drug : drug interactions were analyzed using the Calcusyn software (Biosoft) with a combination index (CI) 〈 1 showing synergism, CI=1 reflecting additivity, and a CI〉1 suggesting antagonism. As an alternative, calculation of the relative risk ratios (RRR) was performed based on the GraphPad software with RRR

Beschreibung: The treatment of pediatric lymphoblastic lymphoma (LL) has developed in parallel with treatment strategies for childhood acute lymphoblastic leukemia using a BFM backbone. The excellent results of the NHL/BFM 90 trial prompted us to design this randomized factorial study to determine whether a regimen without high dose methotrexate (HDMTX) the CCG BFM will result in the same outcome as NHL/BFM-90 and whether intensification with anthracycline and cyclophosphamide would further improve disease free survival. From June 2000 to October 2005, 257 patients with Murphy’s Stage III and IV (excluding CNS disease) LL were randomized to one of the four regimens. All regimens used the BFM/NHL95 backbone. The CCG BFM regimen had intrathecal (IT) methotrexate throughout interim maintenance and maintenance without IV methotrexate. The NHL BFM utilized I.V. Methotrexate 5 Gms/m2 and intrathecal MTX every 2 weeks for four doses during interim maintenance without further intrathecal MTX during maintenance. One of each backbone regimens was further intensified with anthracycline and cyclophosphamide early in induction and delayed intensification. The median age was 10.3 years, 195 (76%) were males; 43 (17%) had 〉5% bone marrow involvement. Twelve patients with CNS disease were not randomized and received intensification and HD HDMTX with delayed CNS radiation (data not reported here). Major toxicities have been related to bone marrow suppression with 4 toxic deaths, 3 due to sepsis and 1 from cerebral hemorrhage. The frequency of grade III/IV neutropenia (alone, with fever or with infection), anemia, and thrombocytopenia were higher in the intensified arms during induction. Three of the four toxic deaths occurred on the intensified arms. The three years EFS of the HDMTX vs. none is 84.5% ± .3.5% vs. 82.7± 3.8 (ρ= 0.93) and the intensification vs. none is 83.4% ±3.7 vs 83.0% ± 3.6 (ρ= 0.66). Therefore, there was no significant difference between treatment arms. These results suggest that neither HDMTX nor early intensification improves EFS in LL. Future direction should focus on identifying biological factors early in therapy so alternative therapies may be investigated.

Beschreibung: Background: A combination of rituximab and CHOP (R-CHOP) is currently the standard treatment for diffuse large B-cell lymphoma (DLBCL). We previously reported the utility of THP-COP regimen consisted of cyclophosphamide (CPA), pirarubicin (tetrahydropyranyl adriamycin: THP), vincristine (VCR), and prednisolone (PSL) in patients with DLBCL (Tsurumi H. et al. Hematol Oncol 2007). THP, a derivative of doxorubicin (DOX), is another anthracycline that has been reported to be less cardiotoxic than DOX. We conducted this phase II study to verify untreated patients with CD20 positive DLBCL. Patients and Methods: The primary objective was to assess the efficacy of R-THP-COP with response rates. Secondary objectives were to assess the overall survival and drug safety. The study was conducted with local ethics committee approval, and all patients gave written, informed consent prior to enrollment. Adverse effects were graded according to the National Cancer Institute Common Toxicity Criteria version 2.0. Over 18 were eligible for inclusion in the study if they had a diagnosis of untreated CD20 positive DLBCL. DLBCL was confirmed by biopsy. All patients had DLBCL at clinical stage (CS) II, III, or IV. Patients aged

Beschreibung: PAX5 is a transcriptional factor playing an important role in B-cell development. Overexpression of PAX5 induced by translocation to the enhancer region of immunoglobulin heavy chain gene occurs in non-Hodgkin lymphomas (NHL), suggesting that PAX5 can be also associated with development of NHL. To identify genes associated with tumorigenesis in malignancies overexpressing PAX5, we performed ChIP-on-chip analysis using PAX5 specific antibody. Non-specifically immunoprecipitated DNA by antibodies can cause false positive results using ChIP-on-chip analysis (background). To reduce the background in ChIP-on chip analysis, we used a dominant negative form of PAX5 and a wild-type PAX5 specific antibody for our ChIP-on-chip analysis. We have previously found a PAX5 chimeric protein expressed in acute lymphoblastic leukemia in which the C-terminal end of PAX5 was replaced by C20ORF112 protein (Kawamata N et al, Proc Natl Acad Sci U S A. Aug. 12, 2008). We have also found that this chimeric protein behaved in a dominant negative fashion over the wild-type PAX5 and suppressed expression of target genes of wild-type PAX5. PAX5 chimeric protein can compete with wild-type PAX5 for binding on the promoter region of direct down-stream target genes. To identify the genes directly regulated by PAX5 in human B-cells, we transfected the dominant-negative form of PAX5 chmeric protein, PAX5-C20ORF112 (PAX5-C20S) into NALM6 human B-cell leukemia cells which constitutively express abundant PAX5. Transfected cells were collected and chromatin immunoprecipitation (ChIP) assay was performed using PAX5 C-terminal specific antibody which can recognize only wild-type PAX5, but not the chimeric PAX5 protein, PAX5C20S. As a control, we also performed ChIP assay using NALM6 cells transfected with an empty vector. Immunoprecipitated DNA was recovered and amplified using the whole genome amplification technique. The DNAs were hybridized with oligonucleotide probes containing the promoter regions of the human genome. The levels of hybridized DNA were quantified and genes directly bound by PAX5 were identified. Comparison between NALM6 cells transfected with the empty vector and PAX5C20S significantly reduced the background and allowed identification of genes directly regulated by PAX5 in NALM6, including BUB1B, SSSCA1, CEP68, and BAG1. BUB1B, CEP68 and SSSCA1 are proteins involved in mitosis; BAG1 is a protein associated with apoptosis. Dysregulation of these genes by overexpressed PAX5 may be associated with development of B-cell malignancies.

Beschreibung: Activation of the Hedgehog (Hh) signalling pathway by loss of function mutations of the Ptch1 receptor promotes stem or progenitor cell proliferation in several cell types, most notably the basal cells of the skin and granule cells of the cerebellum. We have intercrossed MxCre transgenic mice with conditional Ptch1 knockout mice to study the effects of deleting Ptch1 on adult hematopoiesis, with the hypothesis that loss of Ptch1 would activate the Hh pathway leading to increased hematopoietic stem cells (HSC). Within 4 weeks after deletion of Ptch1 with administration of poly(I:C), MxCrePtch1-null mice developed apoptosis of bone marrow pre-B cells and double positive thymocytes. Overall, MxCrePtch1-null mice have 10-fold less pre-B cells and thymocytes. MxCrePtch1-null mice also develop a 3-fold increase in lineage negative c-kit+ Sca-1+ (LKS) bone marrow cells, a cell fraction enriched for HSCs. Despite increased numbers of LKS, loss of Ptch1 did not increase the numbers of HSCs as measured by competitive repopulation assays. MxCrePtch1-null mice also developed typical Ptch1-related tumours including basal cell carcinomas and cerebellar tumours, which was consistent with the ability of the MxCre transgene to delete loxP-flanked genes in cell types other than hematopoietic cells. To determine if the hematopoietic changes observed in the MxCrePtch1-null mice were cell intrinsic or due to loss of Ptch1 on cells of the microenvironment, we intercrossed conditional Ptch1 mice with hematopoietic specific Cre transgenic mice. Surprisingly, HSC-specific deletion of Ptch1 using tamoxifen-inducible SCLert(2)Cre mice did not lead to any increase in LKS numbers. Similarly, lymphoid specific deletion of Ptch1 with the B-cell specific CD19Cretransgene or the T-cell specific LckCre transgene did not lead to any lymphoid defects. The lack phenotype in hematopoietic-specific Ptch1-null mice indicates that Ptch1 is redundant on hematopoietic cells including HSCs. Furthermore, the lack of phenotype also suggests that the defects observed in the MxCrePtch1-null mice were due to loss of Ptch1 in the microenvironment. To prove that Ptch1 regulates the hematopoietic microenvironment, we performed reciprocal transplant experiments whereby lethally irradiated MxCrePtch1- null mice were reconstituted with wild-type bone marrow cells. Remarkably, wild-type hematopoiesis grown within the MxCrePtch1-null microenvironment developed the identical hematopoietic defects with increased LKS and apoptosis of pre-B cells and thymocytes. Conversely, the MxCrePtch1-null hematopoietic defects could be completely rescued by transplant into lethally irradiated wild-type mice. Histological examination of bones from MxCrePtch1-null mice showed marked alterations in trabecular and cortical bone. Given the recent demonstration that the Hh pathway regulates adult bone homeostasis, we hypothesize that the increased LKS and loss of pre-B cells observed in Ptch1-null mice are secondary to changes within the bone marrow cell niche.

Beschreibung: Zinc finger (ZF) transcriptional repressor Gfi-1 plays an important role in hematopoiesis and inner ear development, and also functions as an oncoprotein that cooperates with c-Myc in lymphomagenesis. Gfi-1 represses transcription by directly binding to the consensus DNA sequence in the promoters of its target genes. We report here an alternative mechanism by which Gfi-1 represses CDKN2B encoding the cyclin-dependent kinase inhibitor p15INK4B. Gfi-1 did not directly bind to CDKN2B, but interacted with Miz-1 and, via Miz-1, was recruited to the core promoter of CDKN2B. The C-terminal zinc finger domains of Gfi-1 and Miz-1 are involved in the interaction. Miz-1 is a POZ-ZF transcription factor that has been shown to mediate transcriptional repression by c-Myc. Like c-Myc, upon recruitment to the CDKN2B promoter, Gfi-1 repressed transcriptional activation of CDKN2B by Miz-1 and in response to TGFb. Notably, Gfi-1 and c-Myc formed a ternary complex with Miz-1 and were both recruited to the CDKN2B core promoter via Miz-1, and acted in collaboration to repress CDKN2B. Consistent with its role in repressing CDKN2B transcription, knockdown of Gfi-1 in human leukemic cells resulted in augmented levels of p15INK4B, which was associated with attenuated cell proliferation. The expression of p15INK4B was also significantly higher in Gfi-1−/− mouse bone marrow (BM) cells than in Gfi-1+/+ BM cells. Our data reveal a novel mechanism of transcriptional repression by Gfi-1 and also identify CDKN2B as a new Gfi-1 target gene. The findings may have important implications for understanding the role of Gfi-1 in normal development and the cooperation between Gfi-1 and c-Myc in lymphomagenesis.

Beschreibung: We previously identified the mitochondrial solute carrier, Mitoferrin1 (Slc25a37, Mfrn1) as the principal mitochondrial iron importer essential for heme and iron-sulfur (Fe-S) cluster synthesis in developing erythroblasts. Its closely related paralog, Mitoferrin2 (Slc25a28, Mfrn2) functions in an analogous role in non-erythroid cells. Zebrafish with mutations in Mfrn1 have defects in hemoglobinization and maturation of erythroid cells caused by defective acquisition of iron into the mitochondria (GC Shaw, et al., 2006 Nature 440:96–100). Mfrn1 is highly expressed in embryonic and definitive sites of hematopoiesis in zebrafish and mouse, such as the developing blood island (ICM), fetal liver and adult bone marrow. In contrast, Mfrn2 is ubiquitously expressed, including at very low levels in erythroid cells. To understand the transcriptional regulation of Mfrn1 and Mfrn2, we used bioinformatics tools to identify potential cis-regulatory motifs (CRM) within each gene from mouse. The Gateway-modified Tol-2 vector was used to rapidly clone these conserved, minimal CRM fragments from mouse upstream of the basal promoter and an EGFP-reporter. Each construct was then introduced into zebrafish embryos for transient and stable expression. Using this strategy, we identified CRM’s that recapitulate the endogenous mRNA expression pattern of Mfrn genes during zebrafish development. Germ line stable transmission of the murine Tg(Mfrn1:EGFP) reporter in zebrafish showed robust EGFP expression in erythroid progenitors and mature erythrocytes and the remarkable conservation of function for CRM’s across species. In contrast, the mouse Tg(Mfrn2:EGFP) was expressed in skeletal muscle, heart, liver, and pronephros. The Mfrn1 enhancer is located ~35 kb upstream of the transcription start site and contains two GATA consensus motifs, which bind GATA-1 by chromatin immunoprecipitation analysis. Moreover, the ~150 bp Mfrn1 enhancer fragment exhibits transcriptional activation when coupled to the minimal γ-globin promoter driving expression of a luciferase reporter in K562 cells. Site-directed mutagenesis revealed that both GATA motifs are required for robust erythroid expression. In a complementary approach, transient knockdown of GATA-1 in zebrafish embryos using anti-sense morpholinos selectively ablated Mfrn1 mRNA expression in the ICM, consistent with the epistatic relationship of GATA-1 and Mfrn1. The zebrafish transgenic lines harboring the two murine Mfrn enhancers have proven useful in studying the regulatory and developmental expression in the Mfrn genes in erythroid and non-hematopoietic organs, such as heart and liver. Our results show that the combined use of bioinformatics, Gateway-mediated cloning, and Tol-2 mediated transgenesis in zebrafish embryos is an effective approach to functionally interrogate the transcriptional activity of putative CRM’s in vivo. The conservation and faithful expression of mouse CRM’s in zebrafish demonstrate the utility of this functional approach for analyzing mammalian CRM’s.

Beschreibung: Hematopoietic Stem Cell (HSC) self-renewal and multilineage differentiation potential is governed by multiple intrinsic and extrinsic parameters. Collectively, these parameters dictate the fate of HSC and underscore the heterogeneity observed within phenotypically defined groups of stem cells. While cell cycle status and the genetic profile of HSCs are critical intrinsic modulators of cell fate, interactions with cytokines, growth factors, and cellular elements of the hematopoietic niche (HN) are key extrinsic regulators of stem cell function. We examined the impact of cellular elements of the HN on stem cell fate and maintenance by analyzing the combined effect of calvaria-derived osteoblasts (OB) and mesenchymal stromal cells (MSC) on cultured murine HSC. Murine bone marrow-derived KSL cells were co-cultured with OB alone, MSC alone, or with mixtures of OB and MSC at different ratios for one week. Cultures were supplemented with SCF, Fl-3, Tpo, IL-3, IL-6, IGF1 & OPN. OB alone, maintained the functional properties of cultured HSCs significantly better than MSC thus corroborating the importance of OB in the overall competence of the HN. On day 7, the fold-increase in the number of LSK cells was 1473 ± 291 in OB cultures, 561 ± 159 in MSC cultures, and 603 ± 263 in OB+MSC cultures (n= 4 for all 3 groups). During the same 7 day-period, the number of CFU in progeny cells expanded 74 ± 15 fold in OB cultures, 23 ± 2 fold in MSC cultures, and 27 ± 15 in OB+MSC cultures (n=3 for all groups). The substantial increase in KSL progeny in OB cultures on day 7 was accompanied by a high percentage of cells in active phases of cell cycle (% G0/G1 = 72.5 ± 7.0, n=3) compared to their counterparts in MSC or OB+MSC cultures. In addition, co-culture of KSL cells with OB resulted in an unexpected higher maintenance of the Sca-1+Lin- phenotype (26.5% ± 2.8%) relative to MSC cultures (4.6% ± 1.0%) and OB+MSC cultures (11.7% ± 1.8%; n=3 for all). Only some of these results were reproduced when KSL cells were cultured in OB-conditioned medium suggesting that cell-to-cell contact may be essential for the observed activities. To assess the in vivo potential of LSK cells maintained in these cultures, the 10-day expansion equivalent of 1,000 LSK cells were competitively transplanted in lethally irradiated congenic mice and chimerism was monitored for the next 4 months. At 1 and 2 months post-transplantation, the level of chimerism sustained by LSK cells maintained in OB cultures for 10 days surpassed or was slightly lower than that observed with freshly isolated LSK cells (72.7% vs 59.7% and 57.4% vs 74.7%, respectively) suggesting that OB culture conditions effectively expanded short-term repopulating cells. At 4 months post-transplantation, mice receiving freshly isolated LSK cells were 83.6% ± 1.8% chimeric compared to 53.7% ± 16.1% for mice transplanted with cells from OB cultures and 31.9% ± 21.4% for mice receiving cells from OB+MSC cultures. Overall, these data suggest that OB-LSK interactions promote the maintenance of both short-term and long-term repopulating cells while MSC suppress the OB-mediated activity. To investigate the mechanism of OB-mediated maintenance of stem cell phenotype and function, we examined Notch signaling using Real-Time Q-PCR on cells maintained in culture for 7 days. Relative to the expression in KSL cells, expression of Notch 2 was elevated in OB cultures and suppressed over 2-fold in cultures of MSC and OB+MSC. Similarly, the expression of Jagged 1 and 2, Delta 1 and 4, Hes 1 and 5, Deltex, and SKP2 was increased in OB cultures and suppressed in MSC and OB+MSC cultures. Collectively, these data illustrate that cell-to-cell contact between OB and KSL cells promotes the in vitro maintenance of long-term and short-term repopulating cells and suggest that this stem cell function-promoting activity is induced in part by the upregulation of Notch-mediated signaling between HSCs and osteoblasts. The suppressive effect imparted by MSC on stem cell maintenance compared to cultures of OB alone suggest that these two cellular elements of the HN have opposite effects on the fate and function of stem cells.

Beschreibung: Microenvironment in the stem cell niche plays an important role to regulate self-renewal and differentiation of hematopoietic stem cells (HSCs). We previously showed opposing effects of b-catenin activation on HSC depending on target of activation in the bone marrow microenvironment. To further analyze the microenvironmental regulation of HSC by Wnt/b-catenin signal, we examined b-catenin activation mode in the trabecular bone marrows. In-situ immunohistochemistry of bone marrows revealed a compartmentalized enrichment of b-catenin in the spindle shaped, CD45(−) endosteal stroma of bone marrow (SNO cells) compared to CD45(+) hematopoietic cells. Receptors for canonical Wnt signals (Fz1, 2, 7, 8) or co-receptors (LRP5, 6) were also enriched in the CD45(−) mesenchymal stromal cells of bone marrows than hemapoietic cells. Moreover, accumulation of active form b-catenin was selectively observed in the “stimulated” bone marrows that had been irradiated or injected with Wnt 3a conditioned medium (Wnt 3a-CM), but not in the “steady state” bone marrows. To examine the effect of b-catenin activated stroma on HSCs, 5-FU bone marrow cells were co-cultured in-vitro for 5 days and transplanted into irradiated mice. A 3-fold higher expansion of primitive phenotype (Lin-Sca-1+c-kit+) cells were seen after culture without differences in cell cycle progression. Further, CRU analysis of the transplanted co-cultured cells displayed higher numbers of CRUs regenerated in the recipient bone marrows (65 CRU vs. 1155 CRUs for MIG vs. b-catenin MSC group, respectively). To directly test the effect of b-catenin activated stroma on HSC during normal reconstitution process, we compared HSC self-renewal in the marrows reconstituted with control or b-catenin activated MSCs; MIG or b-catenin transduced MSCs were directly injected into femur with bone marrow cells and each group mice marrows were then mixed (1:1) transplanted into secondary recipient mice for competitive CRU assay. A 3-fold higher CRU frequency was seen for the HSCs derived from marrows reconstituted with b-catenin/MSCs, indicating the physiological significance of b-catenin activation for in-vivo reconstitution. We next investigated the underlying mechanism for stromal b-catenin effects on HSCs. Expression analysis of b-catenin transduced or Wnt3a-stimulated MSCs revealed higher levels of notch ligands (jagged-1, dll-1), which was similarly observed in the trabecular endosteum of mice treated with Wnt3a-CM. A microarray-based expression analysis further supported up-regulation of notch ligands in b-catenin transduced MSCs, as evidenced by induction of dlk-1 and microfibrillar glycoprotein-2, a protein facilitating utilization of jagged-1. Importantly, induction of notch down-stream molecules (Hes-1 and Deltex-1) was demonstrated in the hematopoietic cells (Lin-Sca-1+) cells co-cultured on the b-catenin activated MSCs. Furthermore, enhancing effects of b-catenin/MSC for expanding undifferentiated cells were abrogated by treatment of gamma-secretase 2 inhibitors during the co-culture. These results show that b-catenin activated stromal cells activate notch signal in the contacting HSCs and that activated notch signal underlies the observed stimulatory effects of b-catenin activated stroma on HSCs. Taken together, Wnt/β-catenin activated stroma and the cross-talk with HSCs may function as a physiologically regulated microenvironmental cue for HSC self-renewal in the stem cell niche.

Beschreibung: Signal regulatory protein-α (SIRPA) is an immunoglobulin superfamily transmembrane protein with intracellular docking sites for two Src homology domain containing tyrosine phosphatases, and most abundantly expressed in neurons and myeloid cells. SIRPA is a critical immune inhibitory receptor on macrophages. CD47 is a ligand for SIRPA, and CD47 interaction with SIRPA serves as a ‘self-recognition’ that prevents phagocytosis of the cells expressing CD47. Recently, we (Nature immunology 2007) identified polymorphism in murine Sirpa as a critical modulator of engraftment in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation, and non-obese diabetic (NOD) mouse Sirpa polymorphism has far greater reactivity with human CD47 than that of the respective alleles of other strains resulting in effective engraftment of human hematopoietic cells. In addition, as well as the mouse, human SIRPA is highly polymorphic in the IgV domain. Then, we examined whether polymorphism in SIRPA induced the difference for suppressive effect of human macrophage on hematopoiesis. First, we examined sequence alignment of SIRPA IgV domain (exon 3 of SIRPA) of healthy control 18 people. We identified two major variants; 6 people with variant1 (V1) and 12 people with variant2 (V2). The SIRPA amino acid sequences of V1 and V2 are different in 13 residues, and its residues in orthologous position between species that is polymorphic between NOD and other strains as well as between V2 and V1. Next, we examined that whether there is a difference in the effect on hematopoiesis between V1 macrophage and V2 macrophage by long term culture-initiating cells (LTC-IC) assay on MS-5 stromal cells. For macrophage preparation, peripheral mononuclear cells from healthy donors were purified by positive selection using MACS CD14 Micro Beads (Miltenyi Biotec), and they were incubated with M-CSF for 3 days. For LTC-IC assay, 5×102 differentiated macrophages were seeded onto established MS-5 stroma in 96-well tissue culture plates. The next day, human hematopoietic CD34+ cells were seeded at doses of 102 to 5×103 cells per well and cultured for 4 weeks. At the end of the culture, cells were detached and plated into methylcellulose progenitor assays. Macrophage had a suppressive effect on the number of LTC-IC in all cultures. There was tendency that V1 macrophage had a greater suppression compared to V2 macrophage, however the differences between V1 and V2 were marginal (p=0.03). These data suggests that human macrophages have suppressive effect on hematopoiesis, and human SIRPA polymorphism modulates macrophage-mediated suppression of hematopoiesis in allogenic model, likewise in xenogeneic model of human-to-mouse hematopoietic stem cell transplantation. Moreover, SIRPA polymorphism might be related to graft failure in the allogenic hematopoietic stem cell transplantation.

Beschreibung: In unrelated donor hematopoietic stem cell transplantation (URD-SCT) patients are preferably transplanted with stem cells from a fully HLA matched donor, usually defined as identical for HLA-class I, -DR and -DQ. Since HLA-DPB1 is often not taken into consideration in donor selection, 80–90% of URD-SCTs are mismatched for HLA-DPB1. The role of HLA-DPB1 as transplantation antigen has been unclear, since clinical reports on the impact of matching for HLA-DPB1 on transplant outcome showed conflicting results. HLA-DPB1 mismatching has been associated with an increased risk of graft versus host disease (GVHD). However, we recently demonstrated that HLA-DPB1 specific T cells can mediate a potent graft versus leukemia effect without inducing GVHD. It has been suggested that the controversial effects of matching for HLA-DPB1 in URD-SCT could partly be explained by the assumption that not all HLA-DPB1 differences are immunogenic. This theory was based on the cross-reactive recognition of two HLA-DPB1* 09 specific T cell clones that recognized other HLA-DPB1 alleles sharing amino acids (aa) in position 8–11 of HLA-DPB1 (Zino et al, blood 2004). It was hypothesized that there would be no induction of T cell responses between individuals expressing HLA-DPB1 molecules sharing this aa sequence. This was translated into a classification of permissive and non-permissive HLA-DPB1 mismatches in order to allow a broader donor selection. To investigate whether cross-reactive recognition of other HLA-DPB1 molecules by our previously generated HLA-DPB1*02 or *03 specific CD4+ T cell clones depended on the presence of specific aa sequences we tested recognition of a panel of 14 EBV-LCL expressing 9 different HLA-DPB1 molecules. All HLA-DPB1*02 as well as all *03 specific T cell clones showed cross-reactivity with other HLA-DPB1 alleles and each T cell clone exhibited its own pattern of cross-reactivity. Two HLA-DPB1*0201 specific T cell clones with different TCR-Vβ showed also recognition of EBV-LCL expressing HLA-DPB1*1001 and *1701 or HLA-DPB1*1001, *0901 and *1601 respectively. Five HLA-DPB1*03 reactive T cells clones with different TCR-Vβ showed differential cross-recognition of EBV-LCL expressing HLA-DPB1*0101, *0601, *1101, *1301 and *1401. To identify immunogenic differences the aa sequences of the HLA-DPB1 molecules recognized by the various T cell clones were compared. The HLA-DPB1 molecules recognized by the HLA-DPB1*02 specific T cell clones shared an aa substitution at position 69 compared to the responder cell. However, HLA-DPB1*0601,*0901 and *1901 with the same substitution were not recognized by both T cell clones. This phenomenon was also observed for the HLA-DPB1*03 specific T cell clones, indicating that the cross-reactive recognition of HLA-DPB1 could not be predicted by aa sequences. Next, we analyzed the immunogenicity of various HLA-DPB1 alleles in different stimulator/responder combinations to verify the classification of permissive and non-permissive mismatches. We developed a model to generate allo-HLA-DP responses by transducing HLA-class II negative HELA cells with various HLA-DP molecules and used these cells to stimulate purified CD4+ T cells from HLA-DPB1 homozygous donors. HELA cells transduced with HLA-DPB1*0101, *0201, *0301, *0401, *0402, *0501, *0601, *0901, *1101, *1301, *1401 or *1701 were used as stimulator cells. Responder CD4+ T cells were typed HLA-DPB1* 0201, *0301, *0401 or *0402. 14 days after stimulation, CD4+ T cells were tested for recognition of the stimulator cells and of HELA cells transduced with the responder HLA-DPB1 molecule as a negative control. For these 4 responders, stimulation with 12 different HLA-DP transduced HELA cell lines resulted in specific IFN-γ production in response to the stimulator cells in 47 out of 48 stimulations. 28 CD4+ T cell lines also showed cross-reactive recognition of HELA cells transduced with at least one other HLA-DPB1 molecule. In conclusion, we showed that cross-reactive recognition of various HLA-DPB1 molecules by HLA-DPB1 specific T cells is a common observation. However, we demonstrated that cross-reactivity between HLA-DPB1 molecules by allo-HLA-DPB1 specific T cells does not exclude the generation of immune response between individuals expressing these HLA-DPB1 molecules. By generating multiple allo-HLA-DP specific T cell lines, we showed that all HLA-DPB1 mismatch combinations are immunogenic.

Beschreibung: Patients with mantle cell lymphoma (MCL) and MRD after intensive radiochemotherapy and autologous stem cell transplantation have a high risk of relapse. Allogeneic stem cell transplantation offers the possibility of cure but is associated with a high risk of severe “graft versus host disease”(GvHD). A way to decrease the risk of GvHD while augmenting the “graft versus lymphoma” effect may be the in vitro activation and subsequent transplantation of allogeneic idiotyp-specific T-cells. This study was set out to determine whether cytotoxic T-cell responses specific for peptides derived from the mantle cell idiotype immunoglobulin can be activated in healthy individuals. In four patients with MCL treated in the European Mantle Cell Lymphoma Study Group the immunoglobulin heavy chain (IgH) gene family was amplified in lymphoma samples by PCR and sequenced. Using bioinformatics, the corresponding aminoacid sequence was analyzed for nonapeptides potentially binding to the individual HLA-haplotype. Peptides with a Rammensee-score 〉20 were synthesized. To determine whether these peptides could indeed elicit CD8+ T-cell responses they were used for dendritic cell (DC) pulsation and subsequent T-cell activation. The specificity of the CD8+ T-cells was tested against idiotype-pulsed DC and measured by flow cytometric intracellular interferon (IFN)-gamma staining. The lymphoma specific IgH rearrangements were successfully amplified and sequenced in all patients. In a HLA-A3 positive patient who was in remission after intensive radiochemotherapy and autologous hematopoietic stem cell transplantation three different idiotype HLA-matching peptides with a HLA-A3 binding score 〉20 were predicted from the VH-region, one additional nonapeptide was overlapping to the N-region of the immunoglobulin, rendering this peptide lymphoma-specific. This pool of peptides was synthesized and used for pulsation of monocyte derived dendritic cells (moDC) in two healthy HLA-A3 positive individuals. The maturation of the DC was done according to a standard protocol using proinflammatory cytokines (IL-6, IL-1 beta, TNF-alpha, PGE2). After 2–3 weekly stimulations of lymphocytes that had been depleted of regulatory T-cells 2.1% idiotype-specific CD8+ T-cells were activated in both healthy donors. Interestingly, T-cell stimulation using moDC matured with CD40− and TLR7/8-ligands was more efficient in comparison to the standard protocol and resulted in 12.3% IFN-gamma positive CD8+ cells. In summary, these data suggest, that idiotype-specific T-cells can be activated from healthy individuals by standard lymphocyte stimulating protocols in vitro. Moreover, the ability of moDC to activate idiotype-specific T-cells is exceeded by DC maturation using CD40− in combination with TLR7/8-ligands. These findings may help to improve immunotherapy in the settings of allogeneic transplantation strategies in relapsed MCL patients.

Beschreibung: Ionizing irradiation can cause bone marrow failure leading to death. Effective therapeutic agents capable of promoting or accelerating the recovery of the hematopoietic and/or immune compartment following radiation injury are limited. We and others have previously demonstrated that recombinant human growth hormone promotes hematopoietic and immune recovery following stem cell transplantation and irradiation. Published data suggest that growth hormone elicits its pro-hematopoietic effects via action of insulin-like growth factor 1 (IGF1). Since IGF1 has recently been approved by the Federal Drug Administration to treat other conditions, IGF1 could be brought to the clinic rapidly upon demonstration of its activity. In this study, we sought to determine whether IGF1 has radioprotective activity. The studies were performed using BALB/c mice. Recombinant human insulin-like growth factor 1 (rhIGF1) was administered at a dose of 100 mcg/dose, i.v., once a day, starting within one hour after irradiation. BALB/c mice were irradiated with 7.5 Gy and treated with saline or rhIGF1 for 5 days. In the saline control group, all mice (10 out of 10 mice) died within 25 days following irradiation. By contrast, four out of 10 mice (40%) in the rhIGF1-treated group survived more than 100 days after irradiation (Figure, P

Beschreibung: There are many unanswered questions regarding the potential of stem cells and their ability to contribute to various tissues in the body. One of the difficulties in addressing issues surrounding stem cell transplantation and their contributions to various tissues is their generally poor engraftment under standard transplantation conditions. Also, transplantation via systemic delivery may result in lodging of cells in tissues where they would not normally be present, thus confusing their ability to contribute cells to those tissues. This study seeks to address the engraftment and stem cell delivery method issues by ectopically transplanting intact donor tissue as the source of stem cells and selecting for donor cells using drug resistance to encourage increased engraftment. Donor cell engraftment was followed using flow cytometry to identify eGFP+ cells in the recipient’s PBL. Femur shafts from eGFP transgenic mice, which are also wildtype for the DNA repair gene O6-methylguanine-DNA methyltransferase (MGMT), were removed from donors, stripped of all muscle/fascia and other attached tissues and implanted subcutaneously into MGMT KO mice (also not expressing eGFP). Knockout of the MGMT gene in recipient mice increases the susceptibility of all tissues, especially hematopoietic stem cells to DNA damage from alkylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). There were 4 treatment groups, each with 2–4 immunocompetent, age and sex matched mice. Group A had femurs implanted but did not receive drug treatment, B received a 10mg/kg dose of BCNU (sub-lethal but myelosuppressive) 3 weeks post-transplant, C received a conditioning dose of BCNU 1 week prior to transplant but did not receive a post-transplant selection dose, D received both a conditioning dose and a selection dose. Two weeks post-transplant, regardless of treatment group, only rare but distinct populations of eGFP positive PBL cells (0.01–0.2%) were found. Five weeks post-transplant (2 weeks post-selection dose) there was a slight increase in eGFP positive cells 0.02–0.29% in groups A–C. Of the 3 animals in group D, 2 had a significant increase in eGFP positive PBL cells (from,

Beschreibung: Interaction of CXCR4 expressed on hematopoietic stem and progenitor cells (HSPC) with bone-marrow stromal SDF-1 is believed to play a central role in retention or mobilization of HSPC. Recently, a mobilization regimen of G-CSF was shown to decrease osteoblast number resulting in reduced levels of bone-marrow SDF-1, however the detailed mechanism leading to this reduction is currently unknown. It is unlikely that G-CSF directly regulates osteoblast SDF-1 production since osteoblasts do not express G-CSF receptor. Proteolytic cleavage of SDF-1 by peptidase CD26 in the bone-marrow may be an alternative mechanism responsible for reduction of SDF-1 level. Although CD26 can cleave SDF-1 in vitro, direct evidence of SDF-1 cleavage by CD26 in vivo during G-CSF induced HSPC mobilization has not been demonstrated. We previously demonstrated that neutrophils are required for G-CSF induced HSPC mobilization and that CD26 expression on neutrophils, rather than HSPC, is critical for mobilization. To more fully understand the role of CD26 in altering SDF-1 protein/activity during G-CSF induced HSPC mobilization, we quantitated bone-marrow SDF-1 levels in CD26−/− and wild-type CD26+/+ mice by ELISA during G-CSF administration. A standard 4 day G-CSF mobilization regimen (100 μg/kg bid, sc × 4 days) decreased bone-marrow total SDF-1 from 4.55±0.3 to 0.52±0.06 ng/femur in wild-type CD26+/+ mice (8.7-fold) and from 4.51±0.3 to 0.53±0.05 ng/femur (8.5-fold) in CD26−/− mice. However, despite an equivalent decrease in SDF-1, total CFU mobilization and the absolute number of mobilized SKL cells were decreased (3.1 and 2.0 fold lower, respectively) in CD26−/− mice compared to wild-type CD26+/+ controls. These results suggest that the decrease in total SDF-1 level in marrow seen following G-CSF treatment is independent of CD26. Cytological examination of bone-marrow smears showed that the reduction in SDF-1 levels in bone-marrow of both wild-type CD26+/+ and CD26−/− mice following G-CSF administration correlated with an increase in total absolute bone-marrow neutrophil cell number, suggesting a role for neutrophils in modulation of SDF-1 protein. To determine if neutrophils affect osteoblast SDF-1 production, bone marrow Gr-1+ neutrophils from wild-type CD26+/+ and CD26−/− mice were purified using anti-Ly6G magnetic beads and co-cultured with MC3T3-E1 preosteoblasts in vitro. Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased pre-osteoblast SDF-1 production by similar amounts (15.4-fold vs 14.8-fold respectively), while Gr-1 neg cells from both wild-type CD26+/+ or CD26−/− were without effect on SDF-1 levels. Similarly, Gr-1+ neutrophils from both wild-type and CD26−/− mice decreased SDF-1 produced by MC3T3-E1-derived osteoblasts from 1.85±0.3 to 0.52±0.06 ng/ml (3.5 fold) and 0.56±0.07 ng/ml (3.3 fold) respectively, with Gr-1neg cells having no effect. Gr-1+ neutrophils either from wild-type or CD26−/− mice, but not Gr-1neg cells, significantly induced apoptosis of MC3T3-E1 cells as measured by Annexin-V staining (70.5%±10.2 vs 71.2%±12.5 for wild-type CD26+/+ and CD26−/− neutrophils respectively) and significantly inhibited osteoblast activity (20-fold vs 20.6-fold for CD26+/+ and CD26−/− neutrophils respectively) as measured by osteocalcin expression. Furthermore, irrespective of G-CSF treatment, an inverse correlation between absolute neutrophil number and SDF-1 protein levels was observed, suggesting that G-CSF induces neutrophil expansion but does not directly affect SDF-1 production. Collectively, these results provide additional support for the critical role of neutrophils in G-CSF induced mobilization and strongly suggested that neutrophils directly regulate bone-marrow SDF-1 levels independent of CD26 activity.

Beschreibung: The total nucleated cell count (TNC) is a major factor in the acceptance or rejection of an umbilical cord blood (UCB) unit. Yet it is stem cell potency that will decide whether a UCB unit has the capability to engraft and repopulate the patient after transplantation. The colony-forming cell (CFC) assay has been employed in a retrospective manner in order to document the possibility of growth potential of stem cell products destined for transplantation. Although some centers count and differentiate colonies, many use the assay to document either growth or no-growth because the results are difficult to interpret and provide little predictive value. Subjectivity and lack of an external standard to which the assay can be calibrated and validated means that the CFC assay can neither be employed as a reliable and reproducible stem cell potency assay, nor can it be used to define release criteria of UCB products for transplantation. A cell potency and release assay is required by standards and regulatory organizations. Viability and CD34 may be important for engraftment, but cannot measure stem cell potency. In a recent article in Transfusion (48:620, 2008), Reems et al describe a novel instrument-based, ATP bioluminescence proliferation assay used for UCB and compared at two geographical locations. Results demonstrated a correlation for the assay between the locations with a correlation coefficient of R=0.94 (p

Beschreibung: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation of PIG-A gene in one or few hematopoietic stem cells and subsequent clonal expansion of mutant stem cells that leads to development of symptoms. It is known that PIG-A mutation is insufficient to account for the clonal expansion required for clinical manifestation of PNH. We are proposing a 3-step model of PNH pathogenesis. Step 1 involves the generation of a GPI-deficient hematopoietic stem cell by somatic mutation of the PIG-A gene. Step 2 involves the immunological selection of GPI-deficient hematopoietic stem cells. Based on the close association of PNH with aplastic anemia, it has been suggested that the selection pressure is immune mediated. However, in spite that over 60% of patients with aplastic anemia have subclinical population of GPI-deficient hematopoietic cells at diagnosis, only 10% develop clinical PNH, suggesting that step-1 and 2 are insufficient to cause PNH. Under immune mediated selection pressure, GPI-deficient cells not only survive, but also proliferate much more frequently than usual to compensate for anemia. This elevated proliferation rate increases the chance that additional genetic mutations are acquired, in turn leads to Step 3. Step 3 involves a second somatic mutation that bestows on PIG-A mutant stem cell a proliferative phenotype. According to this hypothesis, we searched for the candidate gene for Step 3. We reported 2 patients with PNH whose PIG-A mutant cells had an acquired rearrangement of chromosome12, making the break within the 3’ untranslated region in HMGA2. This gene encodes an architectural transcription factor which is deregulated in many benign mesenchymal tumors (Blood. 2006 vol.108 no.13, p4232). Recently, many reports show that truncation of 3’ untranslated region of HMGA2 disrupts binding of miRNA, let-7, which regulates both transcription and translation of HMGA2. In fact, these two PNH patients with chromosomal abnormalities had ectopic expression of HMGA2 in the bone marrow. Based on these, we consider HMGA2 as a candidate gene, ectopic expression of which causes proliferation. We have established the method for stable isolation of mRNA and miRNA from blood and bone marrow cells from PNH patients and analyzed the expression of HMGA2 and let-7 by quantitative RT-PCR. We have analyzed the peripheral blood from 8 healthy volunteers and 12 PNH patients. The samples from patients had significantly higher expression of HMGA2 than those from normal volunteers (relative mRNA expression, 4.8±2.4 vs 1.3±0.3, p

Beschreibung: Eperythrozoon ovis, used thought to be a rickettsia but now identified as a mycoplasma, is an erythrocytic agent that causes hemolytic anemia both in animals and human. To demonstrate the epidemiological status of Eperythrozoon ovis infection in Chinese population, 1458 healthy volunteers, 247 patients with hematologic disorders and 106 susceptible people with direct contact to suspected animals were investigated by classical blood smear examination. The positive samples were identified by a specific PCR assay. The microscopic results showed a lot of small organisms attaching on the surface of erythrocytes (Fig 1). The partial 16s rRNA gene of these organisms was amplified using the conserved primers and confirmed as hemoplasma by sequence alignment analysis. Moreover, complement regulatory protein (CR1, CD35), indicating the function of red cell membrane, was tested by flow cytometry with mouse anti-human CD35 and caprine-anti-mouse IgG-FITC reagents. The expression of CR1 might help to elucidate the mechanism why the Eperythrozoon always lead to anemia and icterus in human being (Fig 2). The Eperythrozoon infection rate in healthy was 239/1458, 95/247 in hematologic disease patients and 55/106 in susceptible people, respectively in our results. Results of flow cytometry in peripheral blood sample showed the co-relation between the CD35 expression and the eperythrozoon infection status. The CD35 values increased obviously in the infected cases. In the 4 outbreak cases with FUO (Fever of unknown origin, finally diagnosed as eperythrozoonsis), CD35 reached the peak which might indicate the immuno-defense from the body against the hemotrophic mycoplasma. Gradually, the CD35 value decreased markedly in the long-term infectious cases, which may be explained by anemia and icterus resulting from destroyed membrane of the erythrocyte. Fig. 1: Field emission scanning electron microscope to diagnose the Eperythrozoonsis. The mildly (Fig. 1--1) and severely (Fig. 1–2) infected RBCs. Erythrocyte in picture 1 (Fig. 1--1) had not been deformed but cells in (Fig. 1–2) and (Fig. 1–3) were badly misshapen with some holes on its surface. Transmission electron microscope showed the location of these organisms in erythrocyte depressions (Fig. 1–4) and fibril of these organisms connected with RBC was also observed when these organisms disassociated from erythrocyte (Fig. 1–5). Blood smear stained by Wright-Giemsa mixture demonstrated severe bacteremia (Fig. 1–6). Fig. 1:. Field emission scanning electron microscope to diagnose the Eperythrozoonsis. The mildly (Fig. 1--1) and severely (Fig. 1–2) infected RBCs. Erythrocyte in picture 1 (Fig. 1--1) had not been deformed but cells in (Fig. 1–2) and (Fig. 1–3) were badly misshapen with some holes on its surface. Transmission electron microscope showed the location of these organisms in erythrocyte depressions (Fig. 1–4) and fibril of these organisms connected with RBC was also observed when these organisms disassociated from erythrocyte (Fig. 1–5). Blood smear stained by Wright-Giemsa mixture demonstrated severe bacteremia (Fig. 1–6). Fig. 2: The expression of CD35 antigen on the surface of RBCs was assessed by flow cytometry. Compared to CD35 expression from the controls, no obvious differences were observed from the healthy people(around 4.69) (Fig 2-1). In the positive samples, the CD35 values increase from the negative 4.69 to 10.61 (Fig 2--2). In the outbreak cases, however, a sharp increase of CD35 antigen expression at the RBC external membrane were observed with the values of 28.76(Fig. 2–3 and 2–4), which were significantly higher than the value of the control samples (p

Beschreibung: The production of red blood cells is tightly controlled by the growth factor erythropoietin (EPO). Mice lacking a functional EPO gene die in-utero due to the complete absence of definitive red blood cells. Thus, studying the role of EPO in adult animals has been limited to the use of artificial anemia models such as phenylhydrazine (PHZ) treatment or phlebotomy. In addition, injections of rHuEPO have been shown to be a valuable tool to study erythropoiesis. However, these approaches have limited use as preclinical models since they do not recapitulate an anemia due to EPO deficiency as it is seen in patients with chronic kidney disease (CKD). These patients develop a chronic anemia due to the lack of a sufficient EPO gene expression response. In order to obtain a preclinical model for an EPO deficient anemia we developed a mouse model in which the EPO gene is flanked by two loxP sites (flox allele). In addition, these animals carry the Cre-ERT2 transgene which allows for a tamoxifen controlled activation of the Cre recombinase. Thus, these mice enable the inactivation of the EPO gene in adult animals by tamoxifen administration. After induction of Cre activity, mice heterozygous for the EPO allele (EPOko/flox) develop a moderate to severe anemia within 3 weeks. Although anemic, these mice display only low levels of EPO in their serum. However, within 3 months after induction of the knock-out the anemia resolves. This indicates that the Cre mediated excision of the EPO gene is insufficient and some EPO expressing cells remain. This mimics the clinical situation of patients with CKD. In order to study the response of EPO deficient mice to a severe anemia we treated animals with phenylhydrazine and observed the recovery of these animals. Interestingly, EPO deficient animals, although slightly delayed, fully recover from the induced anemia. Although the measured sEPO levels are significantly lower compared to control animals an increase in splenic erythropoiesis was observed in these animals. This indicates that the hypoxic response triggered by a sudden severe anemia is activating pathways other than EPO that promote erythropoiesis. Taken together, these results show that this novel mouse model will be useful tool as a preclinical model for the anemia associated with CKD. In addition, this model will allow the study of stress erythropoiesis under EPO limiting conditions. The Pfizer Institutional Animal Care and Use Committee reviewed and approved the animal use in these studies. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International

Beschreibung: Romiplostim, a member of the thrombopoietin (TPO) mimetic class, is an Fc-peptide fusion protein (peptibody) that activates intracellular transcriptional pathways leading to increased platelet production via the TPO receptor (also known as cMpl). The peptibody molecule contains two identical single-chain subunits each consisting of human immunoglobulin IgG1 Fc domain, covalently linked at the C-terminus to a peptide containing two TPO receptor-binding domains. Due to the general concern regarding the immunogenic potential for all therapeutic proteins and the specific concern for monitoring antibodies capable of neutralizing thrombopoietin (TPO), an extensive immunogenicity assessment program was developed to support romiplostim. Romiplostim has been engineered to have no amino acid sequence homology to endogenous TPO. A low theoretical risk of developing conformational antibodies that cross-react against TPO exists. This risk was addressed by using an immunogenicity assessment strategy that relied upon a surface plasmon resonance based biosensor immunoassay using the Biacore 3000 capable of simultaneously monitoring antibodies that bind to romiplostim, TPO, or the active peptide portion of romiplostim (TMP). Samples that tested positive for binding antibodies in the Biacore immunoassay were then tested in the definitive functional biological assay to identify any antibodies capable of neutralizing the biological effect of romiplostim or TPO. Serum samples from 236 actively treated subjects were obtained both before and after exposure to romiplostim and were tested for romiplostim and TPO antibodies. In baseline samples, seventeen subjects (7.1%) tested romiplostim antibody positive and 12 subjects (5.1%) tested TPO antibody positive for pre-existing binding antibodies. After romiplostim exposure, twenty-five out of 236 (10.5%) subjects with ITP developed binding antibodies against romiplostim (inclusive of antibodies to both peptide and the whole molecule) and 12 out of 236 (5.1%) subjects with ITP developed binding antibodies against TPO. The antibodies that developed against romiplostim did not cross react with TPO and the antibodies that developed against TPO did not cross react with romiplostim. The incidence of anti-romiplostim neutralizing antibodies among 236 subjects with ITP who were treated with romiplostim across 10 clinical studies was 0.4% (1 out of 236). No cases of anti-TPO neutralizing antibodies were detected in romiplostim treated samples. In conclusion, after thorough immunogenicity assessment of all subjects treated with romiplostim using sensitive methods to detect binding and neutralizing antibodies, only one subject was found positive for the presence of antibodies capable of neutralizing romiplostim that was negative at the time of follow up 4 months later. As expected, none of the subjects treated were positive for antibodies capable of neutralizing TPO. No clinical sequelae were observed in association to the presence of antibodies.

Beschreibung: BACKGROUND: In paroxysmal nocturnal hemoglobinuria (PNH), the lack of the GPI-anchored terminal complement inhibitor CD59 on erythrocytes renders these cells susceptible to continuous complement mediated hemolysis. Hemolysis leads to anemia, fatigue, red blood cell transfusions, renal impairment and increases the risk of thromboembolic events. Urinary iron loss is also common in patients with PNH due to intravascular hemolysis. Eculizumab is a terminal complement inhibitor that has been evaluated in two phase III studies. In previous studies eculizumab significantly reduced intravascular hemolysis (characterized by significant reduction of LDH levels) and significantly reduced transfusion requirements as well as thromboembolic events. In this study we evaluated the change in hemolytic parameters and levels of ferritin parameters in PNH patients treated with eculizumab in two centers (Essen and Homburg/Saar). METHODS: Nineteen PNH patients were treated with eculizumab as follows: 4 x 600mg IV every 7±2 days; 900 mg 7±2 days later; and then 900 mg every 14±2 days. The median therapy duration was 31 months (range 1–40 months). Hemolysis, transfusion requirements and serum iron parameters were analyzed over time of treatment. RESULTS: Eculizumab effectively inhibited intravascular hemolysis in all PNH patients as evidenced by an 79% decrease in LDH levels (mean±SD: 2,110±802 to 349±182 U/l (normal range: 100–247); p=0.0001) and reduced transfusion requirements. Persistent elevation of reticulocytes as well as the reduction of haptoglobin and hemopexin were observed in most patients, which suggest continuing extravascular hemolysis. Interestingly, we observed an 709% increase in ferritin levels in patients treated with eculizumab (median from 73 to 965 μg/l (normal range: 20–290 μg/L); p=0.001). This was more pronounced in patients still requiring some transfusions. Three patients were started on oral iron depletion therapy. No thromboembolic or serious adverse events were observed in eculizumab-treated patients. Two PNH patients were diagnosed with PNH-associated hematological disease (MDS, myelofibrosis). SUMMARY/CONCLUSIONS: Eculizumab is safe and well tolerated in the analyzed cohort of PNH patients. Iron parameters in PNH patients treated with eculizumab should be monitored to determine if iron supplementation should be altered or iron depletion therapy should be considered. While some extravascular hemolysis may persist, intravascular hemolysis is effectively controlled with eculizumab and is associated with a concomitant improvement in anemia and quality of life.

Beschreibung: The Congenital Dyseyrthropoetic Anemias (CDA) are a heterogeneous group of inborn defects characterized by anemia of varying severity and morphological abnormalities of bone marrow erythroblasts. Studying their pathology has the potential to contribute to the understanding of the normal process of erythropoiesis. In CDA type I, the phenotype is largely limited to the red cell lineage with erythroblasts showing characteristic spongy nuclear chromatin on electron microscopy. Its genetic basis is due to missense mutations in the CDAN1 gene (chromosome 15q15), but the biological function of the highly conserved and non-redundant codanin-1 protein remains entirely unknown. We have produced a mouse model for CDA type I by generating transgenic mice from an ES cell line carrying a gene-trap (splice acceptor/β-galactosidase gene/neomycin resistance gene) in intron 25 of the murine CDAN1 gene. Blood and bone marrow from CDAN1gt/wt heterozygotes is morphologically and quantitatively indistinguishable from wild type animals. Northern blot analysis and RT-qPCR of codanin-1 RNA expression confirms a broad pattern of expression with the highest levels seen in erythroid tissue. Flow-cytometric analysis of β-galactosidase activity in erythroid cells from phenylhydrazine treated adult spleens shows the highest levels in the CD71high/Ter119low early erythroblasts, declining with further erythroid maturation. Furthermore, indirect immunofluorescence localizes the codanin-1/β-galactosidase fusion product in the nucleus of the CD71high/Ter119low early erythroblasts, particularly at the interface between euchromatin and heterochromatin. The presence of an in-frame β-gal in the gene trap insert, therefore, provides a method to analyze gene and protein expression from the CDAN1 promoter, and additional distribution data from heterozygous embryos and adults will be presented. Heterozygote crosses produced no homozygotes among liveborn progeny, suggesting embryonic lethality of this state. Analysis of embryos at different stages suggests development of homozygotes ceased at ~6.5d of gestation, prior to the onset of erythropoiesis. These results from the first transgenic mouse model for CDA type I, highlight a non-erythroid role for codanin-1 in early embryonic development, in addition to its role in adult human erythropoiesis. The embryonic lethality of the mouse model is consistent with the absence of homozygote null mutations in the CDAN1 genes analyzed so far in human patients. Creation of mice with the CDA type I phenotype may have to await a knock-in transgenic containing a human mutation, which is currently in preparation. The model described here should be valuable for further studies of the intracellular localization of codanin-1 and the identification of any relevant protein binding partners.