ALBERT

Description: Design/methods: Rituximab added to 8 cycles of CVP (R-CVP) chemotherapy improves time to progression and duration of response in previously untreated patients with stage III/IV CD20 positive follicular NHL compared with CVP alone (Marcus et al Blood2005;105:1417–23). A protocol pre-planned analysis of this study with a median follow-up of 53 months has now been performed. Results: A total of 321 patients (median age 53 years) were recruited. Eighty-three percent of patients in both arms had intermediate to high-risk disease according to the Follicular Lymphoma International Prognostic Index (FLIPI, score 2–5). Median time to progression or death (TTP) in the R-CVP arm was 34 months compared with 15 months in the CVP arm, p

Description: Introduction: Kidney transplantation is the treatment of choice in patients with end-stage renal failure. However, the immunosuppression has severe side effects, such as chronic allograft nephropathy, cardiovascular morbidity and neoplasia. An additional effect of inhibiting mTOR is microcytic anemia. We have explored the pathophysiology of this effect. Methods: Erythroid progenitor cells were isolated from peripheral blood of healthy control persons (HC; n=8), or kidney transplant recipients with chronic sirolimus treatment with (SRL+MC; n=8) or without (SRL-MC; n=8) microcytosis. The isolated progenitor cells were then cultured in a semi-solid medium, containing 3 U/ml erythropoietin, in the absence or presence of SRL (5ng/mL) for 14 days. Burst forming unit erythroid (BFU-E) derived colonies were then counted through an inverted microscope considering that each colony consists of more than 40 cells. Cultures were performed in duplicate and colonies were counted in the entire culture dish. Results: Hemoglobin was 13.17 (SRL+MC) and 13.23 (SRL-MC) g/dL. RBC count was 5.22 and 4.55x106 /μL in SRL+MC and SRL-MC respectively. MCV was 76 in SRL+MC and 87 fL in SRL-MC. Presence of SRL in the culture medium led to a decrease in the number of colonies in healthy controls and kidney transplant patients (43.2±4.5 vs. 28.5±3.6 BFU-E derived colonies, Mean±SEM, p

Description: Introduction Tangeretin, a polymethoxyflavon present in citrus peel oil, was found to inhibit Erk-phosphorylation in T47-D breast cancer cells, induce cell death and block invasion in the chick heart invasion assay. Erk-phosphorylation has been implicated in the growth of bcr-abl transformed cell lines as a result of constitutive abl-activation. Here we investigate whether tangeretin can induce apoptosis and growth arrest in the bcr-abl+ erytroleukemia cell line K562, how it affects signal transduction pathways and the balance between pro- and anti-apoptotic proteins. Methods Tangeretin was dissolved in DMSO and used at concentrations up to 100 μM. K562 cells were cultured in RPMI 1640–10% FBS in vitro. Proliferation was followed by MTT test. Apoptosis, cell cycle analysis and bcl-2 expression was assessed by flow cytometry. PARP, Erk1/2, p38, Akt, JNK, p70S6K, and P-p38, P-Akt, P-JNK, and P-p70S6K, bcl-XL and mcl-1 were assessed by Western Blot. Results Tangeretin shows a time and concentration dependent effect on bcr-abl + K562 cells with a LD50 of 50–70 μM. This effect is accompanied by a G2/M arrest and a significant increase in the percentage of subG0 cells and PARP cleavage at 24 hrs. In short term kinetics (30 minutes) tangeretin inhibits the phosphorylation of Erk. No effect on total Erk1/2, p38, Akt, JNK, p70S6K, and P-p38, P-Akt, P-JNK, and P-p70S6K could be observed. After 24 and 48 hours of treatment, tangeretin is capable of stimulating the phosphorylation of Erk and p70S6K. At the same time activation of procaspase-3 and -9 and downregulation of the anti-apoptotic proteins Mcl-1 and Bcl-xL were seen. Bcl-2 expression was analysed by flow cytometry and was also downregulated. Conclusion The citrus flavonoid tangeretin is capable of inducing apoptosis and growth arrest in bcr-abl positive K562 cells through activation of caspase-3 and -9 accompanied by a biphasic change in phosphorylation of Erk and p70S6K.

Description: One of the key events during hemostasis is the conversion of the zymogen factor X (FX) into its enzymatically active form FXa by the protease factor IXa (FIXa). FIXa assembles on a phospholipid surface together with its cofactor factor VIIIa (FVIIIa) which transforms FIXa from a very poor enzyme into a highly active protease. In order to mimic the stimulating effect of coagulation FVIIIa on FIXa, we identified a series of antibodies specific for FIX that exhibit FVIII-like activity. Upon binding to human FIXa these antibodies increased the turnover number (kcat) of FIXa-catalysed FX activation approximately 10-fold. Procoagulant activity of these anti-FIXa antibodies could be detected in model systems containing purified proteins as well as in FVIII-depleted plasma and FVIII-inhibitor plasma. In plasma-assays contact activators and tissue factor were applied as a trigger, and the antibodies were effective in both cases. Our findings demonstrate that FVIII can be at least partially replaced by an antibody which might open up a new strategy for improving the treatment of hemophilia.

Description: The signal transducer and activator of transcription 5 (STAT5) has recently been implicated as essential pro-oncogenic factor in the pathogenesis of myeloid leukemias in mice (Cancer Cell2005;7:87–99). More recently, STAT5 activation has also been described to occur in human leukemias. However, so far, little is known about the expression of activated/tyrosine phosphorylated STAT5 (pSTAT5) in various myeloid neoplasms and about the distribution of pSTAT5 in the cellular compartments of the normal and leukemic bone marrow (bm). We have examined the expression of pSTAT5 in the bm in patients with acute myeloid leukemia (AML, FAB M0, n=3, M1, n=6, M2, n=4, M3, n=5, M4, n=5, M5, n=4, M6, n=5, M7, n=4), chronic myeloid leukemia (CML, chronic phase, n=4, accelerated phase, n=5, blast phase, n=5), and systemic mastocytosis (SM, n=30), as well as in the normal bm (n=5). Expression of pSTAT5 was determined on paraffin-embedded bm sections by immunohistochemistry using the pSTAT5-specific antibody AX1. In the normal bm, the antibody AX1 was found to react with megakaryocytes and immature myeloid progenitor cells, whereas erythroid cells and mature granulocytic cells did not stain positive for AX1. In patients with AML and CML, the distribution of pSTAT5 showed a similar pattern. In fact, pSTAT5 was found to be expressed in leukemic blast cells without differences among FAB types as well as megakaryocytic cells, but not in erythroid cells. In patients with SM, neoplastic mast cells were found to be immunoreactive for pSTAT5. Interestingly, in all patients and all cells examined, pSTAT5 was found to be localized in the cytoplasm rather than in the nucleus. The cytoplasmic distribution of pSTAT5 in neoplastic cells was confirmed by immunocytochemical staining experiments performed on primary isolated neoplastic cells (AML, CML, mastocytosis) and respective cell lines (U937, KG1, K562, KU812, HMC-1). In each case, the reactivity of neoplastic cells with the AX1 antibody was abrogated by preincubation of the antibody with a pSTAT5-specific blocking peptide. Moreover, the expression of cytoplasmic pSTAT5 in the leukemic cell lines was demonstrable by flow cytometry. To study the molecular mechanisms underlying STAT5-activation in neoplastic cells, Ba/F3 cells with doxycycline-inducible expression of disease-specific oncoproteins, namely BCR/ABL (CML) and KIT-D816V (SM) were employed. Induction of these oncoproteins in Ba/F3 cells resulted in massive activation of pSTAT5 and DNA binding activity as shown by EMSA and supershift assays. In summary, our data show that neoplastic cells in AML, CML, and SM express cytoplasmic pSTAT5, and that disease-related oncoproteins contribute to STAT5-activation. The particular cytoplasmic localization of pSTAT5 in neoplastic cells suggests that apart from its function as a transcription factor, pSTAT5 may have an additional role as a cytoplasmic regulator in these malignancies.

Description: Zap-70 is accepted as a surrogate marker for mutational status of immunoglobulin heavy-chain variable region genes in B-CLL. Whether Zap-70 is a functional target for treatment of the more aggressive CLL cells that express unmutated IgVH is still under investigation. The use of methylprednisolone (Mp) is widespread in the treatment of B-CLL related concomitant auto-immune disorders. We have recently demonstrated that Zap-70+ B-CLL cells are more resistant to chemotherapy but more susceptible to apoptotic cell death by steroids than Zap-70 negative cells in vitro. We investigated the possible influence of Mp on the expression of Zap-70 in B-CLL cells. Mononuclear cells of 15 patients with B-CLL were isolated by Ficoll centrifugation and incubated with different concentrations of Mp (range 0.1–500 μM). After 24h and 48h, Zap-70 expression, phospho-ZAP-70 (Tyr 319 and Tyr 493) and apoptosis were evaluated by flow cytometry (indirect staining ZAP-70 and phospho-ZAP-70 and Annexin V-FITC/propidium iodide double staining for apoptosis) and Western blotting when appropriate. Student’s t-test was used for statistical analysis. Mp induces time and dose dependent apoptosis in B-CLL cells (LD50 48h=16.8 μM). After 24h and 48h of incubation at doses of 50 μM, Zap-70 expression decreased significantly (50 μM: p

Description: Hemophilia A is a lead candidate for treatment by gene therapy because small increments in the missing secreted protein product, coagulation factor VIII (FVIII), would result in substantial clinical amelioration. Clinically relevant therapy might be achieved by stably delivering a human FVIII cDNA to correct the bleeding disorder. We used the Sleeping Beauty (SB) transposon, delivered as naked plasmid DNA by tail-vein injection, to integrate B-domain–deleted FVIII genes into the chromosomes of hemophilia A mice and correct the phenotype. Since FVIII protein is a neoantigen to these mice, sustaining therapeutic plasma FVIII levels was problematic due to inhibitory antibody production. We circumvented this problem by tolerizing 82% of neonates by a single facial-vein injection of recombinant FVIII within 24 hours of birth (the remaining 18% formed inhibitors). Achievement of high-level (10%-100% of normal) FVIII expression and phenotypic correction required co-injection of an SB transposase-expressing plasmid to facilitate transgene integration in immunotolerized animals. Linker-mediated polymerase chain reaction was used to clone FVIII transposon insertion sites from liver genomic DNA, providing molecular evidence of transposition. Thus, SB provides a nonviral means for sustained FVIII gene delivery in a mouse model of hemophilia A if the immune response is prevented.

Description: Design/Methods: We recently demonstrated in a phase III trial that the addition of rituximab to each of 8 cycles of CVP (R-CVP) chemotherapy significantly improves the clinical outcome of previously untreated patients with stage III/IV CD20 positive follicular NHL when compared to CVP alone (Marcus et al., Blood2005; 105: 1417–23). A multivariate Cox regression analysis of time to progression or death (TTP) showed a treatment benefit in all patient subgroups according to baseline risk factors, except for patients with a baseline hemoglobin level below normal. We now present an updated analysis of all major trial endpoints with 42 months follow-up (FU). Results: A total of 321 patients (median age 53 years) were recruited (159 CVP, 162 R-CVP). Approximately half of the patients had high-risk disease according to the Follicular Lymphoma International Prognostic Index (FLIPI, score 3–5). The median TTP was more than doubled for patients receiving R-CVP compared to CVP alone (33.6 months vs 14.5 months, p

Description: Abstract 2679 Poster Board II-655 Introduction: Bendamustine, a hybrid alkylating agent, shows a unique mechanism of action with a good toxicity profile in various hematological malignancies, particularly in non-Hodgkin's lymphoma. Bendamustine (B) in combination with Rituximab (R) was demonstrated to be an effective regimen in first- or second-line treatment of patients (pts) with indolent lymphomas. So far, however, no data about the capacity of peripheral blood stem cell (PBSC) mobilization or a possible stem cell toxicity of B has been studied. It is still an open and clinically relevant question, if pts treated with B are able to mobilize enough stem cells for subsequent high dose therapy with autologous stem cell support. Objective: We performed a prospective randomized multicenter phase-III trial to compare the efficacy and safety of the combination of Bendamustine plus Rituximab (B-R) versus CHOP plus Rituximab (CHOP-R) as first-line therapy for follicular, indolent and mantle cell lymphomas. Chemotherapy was given in 6 cycles each (B: 90 mg/m2 d1+2). One of the secondary objectives (not mandatory) of this study was to evaluate the possibility to mobilize a sufficient number (at least 2.0 × 10E6 CD 34+ cells/kg) of PBSC and progenitor cells in younger pts after completing either B-R or CHOP-R and to compare both stem cells yields. Results: 549 pts have been randomized, 513 pts (B-R = 260, CHOP-R = 253) are evaluable. In each arm, 23 PBSC mobilizations have been performed. In the B-R as well as in the CHOP-R arm each 18 mobilizations were performed directly after completion of therapy, and in 5 pts stem cells were collected at the time of first relapse. Patient characteristics: initial bone marrow involvement was observed in 17 of 23 pts in the B-R arm and 14 of 23 pts in the CHOP-R arm, respectively. Median patient age was 51 years (B-R) and 53 years (CHOP-R). The mobilization regimen was either high dose cyclophosphamide + G-CSF (9 pts in each arm) or G-CSF alone in 7 pts after B-R and in 2 pts after CHOP-R, respectively. Alternative regimens such as Dexa-BEAM, DHAP, ICE and others were also used in the remaining patients. The median CD34+ cell-count/kg was not significantly different in the two arms with 4.55 × 10E6 CD34+ cells/kg (range 1.68 – 12.35) in the B-R group and 6.17 × 10E6 CD 34+ cells/kg (range 1.68 – 20.39) in the CHOP-R group, respectively. In both arms the medium number of apheresis to achieve these yields was not different (B-R: 1.85 vs. CHOP-R: 1.66). Only 3 pts were not able to mobilize at least 2.0 × 10E6 CD 34+ cells/kg: 1 patient after B-R (1.68 × 10E6 CD 34+ cells/kg), and 2 pts after CHOP-R (1.68 × 10E6 CD 34+ cells/kg, and in 1 patient no mobilization was possible). In patients who were mobilized directly after completion of first-line chemotherapy (18 pts in each arm), again, no differences (B-R: median 5.52 × 10E6 CD 34+ cells/kg vs. CHOP-R: median 7.35 × 10E6 CD 34+ cells/kg) were observed. The yield of stem cells in each 5 pts of both arms who were mobilized at the time of first relapse was also nearly the same (B-R: median 8.79 × 10E6 CD 34+ cells/kg vs. CHOP-R: median 7.3 × 10E6 CD 34+ cells/kg). Conclusions: Our results demonstrate that the collection of sufficient numbers of PBSC after B-R treatment is possible and appears to be comparable to the PBSC yield after prior treatment with CHOP-R. Disclosures: Rummel: Roche Pharma AG: Honoraria, Research Funding; Mundipharma: Honoraria, Research Funding.

Description: The endothelial receptors that control leukocyte transmigration in the postischemic liver are not identified. We investigated the role of junctional adhesion molecule-A (JAM-A), a receptor expressed in endothelial tight junctions, leukocytes, and platelets, for leukocyte transmigration during hepatic ischemia-reperfusion (I/R) in vivo. We show that JAM-A is up-regulated in hepatic venular endothelium during reperfusion. I/R-induced neutrophil transmigration was attenuated in both JAM-A-/- and endothelial JAM-A-/- mice as well as in mice treated with an anti-JAM-A antibody, whereas transmigration of T cells was JAM-A independent. Postischemic leukocyte rolling remained unaffected in JAM-A-/- and endothelial JAM-A-/- mice, whereas intravascular leukocyte adherence was increased. The extent of interactions of JAM-A-/- platelets with the postischemic endothelium was comparable with that of JAM-A+/+ platelets. The I/R-induced increase in the activity of alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and sinusoidal perfusion failure was not reduced in JAM-A-/- mice, while the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive hepatocytes was significantly higher. Thus, we show for the first time that JAM-A is up-regulated in hepatic venules and serves as an endothelial receptor of neutrophil transmigration, but it does not mediate leukocyte rolling, adhesion, or platelet-endothelial cell interactions. JAM-A deficiency does not reduce I/R-induced microvascular and hepatocellular necrotic injury, but increases hepatocyte apoptosis, despite attenuation of neutrophil infiltration. (Blood. 2005;106:725-733)