ALBERT

Beschreibung: CD40 and CD20 are expressed in several B-cell malignancies and represent attractive targets for therapeutic intervention. The anti-CD20 monoclonal antibody, rituximab, is an approved drug for the treatment of non-Hodgkin’s lymphomas. however, the existence of patients with rituximab-resistant disease limits its clinical utility. We have previously reported that the novel, highly potent, fully human antagonistic anti-CD40 monoclonal antibody, CHIR-12.12, generated from XenoMouse® mice (Abgenix, Inc) has greater anti-tumor activity than rituximab in both rituximab-responsive and rituximab-resistant human NHL models. In this study, we evaluated the potential therapeutic application of combining CHIR-12.12 and rituximab for the treatment of NHL. Namalwa is a Burkitt’s lymphoma cell line that gives rise to aggressive rituximab-resistant tumors when implanted in nude mice. Direct treatment of these tumor cells with CHIR-12.12 or rituximab in culture does not affect cell growth when compared to treatment with an isotype control antibody. Although Namalwa cells express more CD20 than CD40 (average of 10,059 CD20 and 3,138 CD40 molecules per cell respectively, P=0.05), when tested for in vitro ADCC killing using human NK cells as effectors, CHIR-12.12 mediated stronger target cell lysis than rituximab (31.43% vs. 14.15%, P

Beschreibung: A strong link between asparaginase-containing (ase +) chemotherapy regimens and venous thrombotic events (VTE) has been established for pediatric acute lymphoblastic leukemia (ALL), but this association has not been studied in adult ALL. We recently adopted as our first-line therapy for adult ALL a treatment protocol (DFCI 00–01) that employs weekly high-dose ase throughout a 27 week intensification phase. We reviewed the records of 92 adult patients consecutively accrued at Princess Margaret Hospital (from Jan 2000 to Dec 2003) for venous thrombotic events (VTE). Median age at diagnosis of ALL was 41±17 y, or 19±3 y for those who presented after a history of pediatric ALL (n=6), and 42±16 y for adults who presented with de novo ALL (n=86). Thirty-five (37%) were female, 59 (63%) male. Twenty-four (26%) were Philadelphia-chromosome positive; 71% of these received regimens containing imatinib. At least 1 symptomatic, radiologically confirmed VTE was seen in 19 patients (21%), with 3 (3%) diagnosed prior to any antileukemic therapy. Overall survival (OS) versus thrombosis-free survival (TFS) is illustrated in the Kaplan-Meier curve below. Median time to VTE was 94±92 d [95% CI]. Average age of patients with VTE was 44 ±15y; no events were observed in patients with a prior pediatric history of ALL. The sites of VTE were: 10/22 (46%) in the lower extremities, 5/22 (23%) propagating from central venous access catheters (CVAC), 4/22 (18%) as pulmonary emboli (2 in isolation), and 3/22 (14%) CNS (2 cerebral venous sinus and 1 retinal venous thrombus). The first antileukemic regimen administered was DFCI 00–01 ± imatinib in 79/92 (86%), HyperCVAD ± imatinib in 7 (8%), an unclassified ase+ regimen in 1 (1%), or an unclassified asparaginase-excluding (ase−) regimen in 5 (5%). A significant difference in the rate of ase-dependent VTE was observed when all regimens (1° or subsequent) were counted, with 18 VTE during 103 ase+ regimens (17.5%), versus only 1 VTE in 32 ase− regimens (3.1%), [p=0.04]. Median time to VTE in DFCI 00-01was 109±98d, most often occurring during the intensification phase. The odds ratio for VTE with any T-cell subtype of ALL compared to any B-cell subtype was 4.6 [p=0.0043]. Conclusions : A high, significant VTE rate (~18%) was observed in adults with ALL undergoing ase+ antileukemic therapy, nearly 6X the rate observed either prior to treatment or during ase− regimens (~3%). Ase is thus strongly associated with VTE, warranting improved surveillance and antithrombotic prophylaxis. Risk factors (underlying thrombophilias, leukemic subtypes) similarly deserve further study. Figure Figure

Beschreibung: We investigated the relevance of p53 deletions to the clinical outcome of multiple myeloma (MM) patients treated with high-dose chemotherapy and autologous stem cell transplantation. Hemizygous p53 gene deletions were detected by cytoplasmic Ig-enhanced interphase fluorescence in situ hybridization (cIg-FISH) in 10 of 105 (9.5%) patients studied. p53 deletions were associated with higher serum calcium (p=0.0062) and creatinine (p=0.013) levels but there were no association with patient age, gender, β-2 microglobulin, C-reactive protein, hemoglobin, albumin, bone lytic lesions, or immunoglobulin isotype. There were no association of p53 deletions with chromosome 13q deletions, translocation t(11;14) or t(4;14). The overall response rates were similar in patients with and without p53 deletions (67% vs 71%). However, patients with p53 deletions had significantly shorter progression free (median 7.9 vs. 25.7 months, p=0.0324) and overall survival (median 14.7 vs. 48.1 months, p=0.0008) than patients without a p53 deletion. A multivariate analysis confirmed p53 deletion was an independent prognostic factor predicting shortened progression free (p=0.0009) or overall survival (p=0.0002) in myeloma patients after high-dose chemotherapy and autologous stem cell transplantation.

Beschreibung: B-cell chronic lymphocytic leukemia (CLL) is characterized by in vivo accumulation of long-lived CD5+ B cells. However when cultured in vitro CLL cells die quickly by apoptosis. Protection from apoptosis in vivo is believed to result from supply of survival signals provided by cells in the microenvironment. We and others have previously reported that CLL cells express CD40 receptor, and that CD40 stimulation of CLL cells may rescue CLL cells from spontaneous and drug-induced apoptosis in vitro. These observations suggested that blocking CD40-CD40L pathway might deprive CLL cells from survival signals and induce apoptosis. To test this hypothesis, we have generated a fully human anti-CD40 blocking monoclonal antibody in XenoMousemice (Abgenix, Inc.). The antibody CHIR-12.12 was first evaluated for its effect on normal human lymphocytes. Lymphocytes from all 10 healthy blood donors did not proliferate in response to CHIR-12.12 at any concentration tested (0.0001 mg/ml to 10 mg/ml range). In contrast, activating CD40 on normal B-lymphocytes by CD40L induced their proliferation in vitro. Importantly, CHIR-12.12 inhibited CD40L- induced proliferation in a dose dependent manner with an average IC50 of 51 ± 26 pM (n=10 blood donors). The antagonistic activity of CHIR-12.12 was then tested in primary CLL samples from 9 patients. CHIR-12.12 alone did not induce CLL cell proliferation. In contrast, primary CLL cells incubated with CD40L, either resisted spontaneous cell death or proliferated. This effect was reversed by co-incubation with CHIR-12.12 antibody, restoring CLL cell death (n=9). CHIR-12.12 was then examined for its ability to lyse CLL cell line EHEB by antibody dependent cell mediated cytotoxicity (ADCC). Freshly isolated human NK cells from normal volunteer blood donors were used as effector cells. CHIR-12.12 showed lysis activity in a dose dependent manner and produced maximum lysis levels at 0.1 mg/ml. When compared with rituximab, CHIR-12.12 mediated greater maximum specific lysis (27.2 % Vs 16.2 %, p= 0.007). The greater ADCC by CHIR-12.12 was not due to higher density of CD40 molecules on CLL cell line compared to CD20 molecules. The CLL target cells expressed 509053 ±13560 CD20 molecules compared to 48416 ± 584 CD40 molecules. Collectively, these preclinical data suggest that CHIR-12.12 monoclonal antibody may have a therapeutic role in patients with CLL.

Beschreibung: Background: Inability to collect adequate numbers of PBSCs remains an obstacle to curative ASCT in heavily pretreated patients with relapsed or refractory lymphoma and other malignancies. Retrospective data from our institution have shown that pts with greater exposure to marrow toxic agents have only a 50% rate of successful stem cell collection versus 87% in pts with lesser exposure. We identified heavily pretreated patients at high risk of mobilization failure as rated by a previously validated chemotherapy scoring system (Drake et al. Br J Hematol, 1997;98:745) and used a more intensive regimen of SCF, G-CSF and CY for first-line stem cell mobilization. Methods: Pts with acute myeloid leukemia (AML) or relapsed/refractory Hodgkin’s disease (HD) or non-Hodgkin’s lymphoma (NHL) with a chemotherapy score 〉42 were eligible. Following completion of salvage or consolidation chemotherapy, consenting patients were mobilized with CY 2.5 g/m2(day 1), SCF 20 ug/kg/day and G-CSF 10 ug/kg/day, both starting day 4, and continuing until completion of leukapheresis. PBSC collection began day 11 (if PB CD34 cells were 〉5/mL) and continued to a target of 2 x 106 CD34+ cells/kg or for a maximum of 5 days. Results: Twenty-seven pts were enrolled. Diagnoses: NHL n=22; HD n=1; AML n=4. M:F ratio = 14:13; median age 52 yrs (range 17–63); time from diagnosis to study entry, median 18 mos (range 5–70 mos). Nineteen of 23 lymphoma pts received 6–8 cycles of CHOP/ABVD followed by salvage chemotherapy. The other 4 pts had transformed lymphoma and received CVP or chlorambucil prior to transformation. Sixteen pts responded after 1 salvage regimen; 7 pts required 2 salvage regimens [ESHAP, GDP, DHAP, miniBEAM, or CHOP (for transformed pts)]. AML pts were in CR at time of study entry and had received ≥2 consolidation cycles with high-dose cytarabine. Sixteen of 27 pts (59%)[95% CI 44–78%] were successfully collected (median of 3.21x 106 CD34+ cells/kg (range 2.21–14.41) in a median of 1 apheresis (range 1–5; 11 were collected with a single apheresis). However, 3 of the 4 AML pts did not mobilize. Neutrophils

Beschreibung: The identification of genes affecting plasma concentrations of biological traits remains difficult, as the loci affecting such traits (termed quantitative trait loci) tend to explain only a fraction of the phenotypic variation. Evidence on inter-relation (i.e. clustering) of coagulation factors in the literature (Van Hylckama Vlieg 2003) suggests the existence of quantitative trait loci, which influence plasma concentrations of several quantitative traits (i.e.have a pleiotropic effect) outside the genes coding for these factors. The aim of the present study was to identify clusters of pro- and anticoagulant factors within a large protein C deficient kindred using principal components analysis. In addition, we wanted to determine how much of the variance within these clusters could be attributed to the genetic variation within a single large pedigree. Levels of the following analytes were measured in family members: prothrombin, factor V, VII, VIII, IX, X, fibrinogen, von Willebrand factor, antithrombin, protein C and protein S. Subjects with the 3363C protein C mutation, a personal history of thrombosis or those using oral anticoagulants, and women pregnant at the time of the blood draw were excluded from the analyses. To identify clusters of haemostatic factors, the principal component method with orthogonal varimax rotation was performed using SPSS. We used a factor loading of 〉0.40 as a marginal value to include coagulation factors in a cluster. Heritability, the proportion of the phenotypic variance attributed to polygenes, and common household effect, the proportion of the variance attributed to environmental factors shared within a household, were estimated for each principal component score with an eigenvalue (the variance attributable to a particular principal component) greater than or equal to 1 using the variance component method in SOLAR (Almasy & Blangero 1998). The distribution of each score was assumed to be multivariate normal with a variance-covariance matrix following the formula: covariance (one person to another person)=h2K + c2H + e2I, with K derived from the kinship matrix, H from the household matrix and I from the identity matrix. The additive genetic and household components of variance were estimated using maximum likelihood analysis. A total of 87 family members met the inclusion criteria. The principal components analysis identified 3 components which explained 60% of the variance: component 1 included all vitamin K dependent factors (prothrombin, factor VII, factor IX and factor X, protein C and protein S), component 2 consisted of factor V, factor IX, fibrinogen and antithrombin, which all can interact directly with thrombin, and component 3 consisted of factor VIII and its carrier protein von Willebrand factor. The heritability estimates for these 3 components were, respectively, 96% (p=0.002), 87% (p

Beschreibung: Platelet factor 4 (PF4), an abundant platelet α-granule protein, accelerates in vitro generation of activated protein C (APC) by soluble thrombin/thrombomodulin (TM) complexes up to 25-fold. To test the hypothesis that PF4 similarly stimulates endothelium-associated TM, we assessed the influence of human PF4 on thrombin-dependent APC generation by cultured endothelial monolayers. APC generated in the presence of 1 to 100 μg PF4 was up to 5-fold higher than baseline for human umbilical vein endothelial cells, 10-fold higher for microvascular endothelial cells, and unaltered for blood outgrowth endothelial cells. In an in vivo model, cynomolgus monkeys (n = 6, each serving as its own control) were infused with either PF4 (7.5 mg/kg) or vehicle buffer, then with human thrombin (1.0 μg/kg/min) for 10 minutes. Circulating APC levels (baseline 3 ng/mL) peaked at 10 minutes, when PF4-treated and vehicle-treated animals had APC levels of 67 ± 5 ng/mL and 39 ± 2 ng/mL, respectively (P 〈 .001). The activated partial thromboplastin time (APTT; baseline, 28 seconds) increased maximally by 27 ± 6 seconds in PF4-treated animals and by 9 ± 1 seconds in control animals at 30 minutes (P 〈 .001). PF4-dependent increases in circulating APC and APTT persisted more than 2-fold greater than that of control's from 10 through 120 minutes (P ≤ .04). All APTT prolongations were essentially reversed by monoclonal antibody C3, which blocks APC activity. Thus, physiologically relevant concentrations of PF4 stimulate thrombin-dependent APC generation both in vitro by cultured endothelial cells and in vivo in a primate thrombin infusion model. These findings suggest that PF4 may play a previously unsuspected physiologic role in enhancing APC generation. (Blood. 2003;102:146-151)

Beschreibung: Plasminogen (Pg) is the zymogen of the fibrinolytic enzyme plasmin (Pn). Fibrin (Fn) promotes the activation of Pg by tissue-type plasminogen activator (t-PA) by serving as a template that brings t-PA and Pg into close proximity. In addition, proteolysis of Fn by Pn generates carboxyl-terminal lysine residues that provide nascent high affinity binding sites for Glu-Pg, thereby promoting Pg activation. Pg activation is also enhanced when Glu-Pg is converted to Lys-Pg, a derivative with higher Fn affinity. Therefore, kinetic and functional data suggest that Pg binding to Fn is a key aspect of efficient Pg activation. To explore this concept, we performed binding and kinetic analyses with Mini-Pg, an elastase-derived fragment of Glu-Pg with reduced Fn affinity. Binding of Glu-Pg, Lys-Pg and Mini-Pg to immobilized fibrinogen (Fg) and fibrin monomer (Fm) was monitored by surface plasmon resonance. The affinity of Glu-Pg for Fm is 4-fold higher than that for Fg with Kd values of 3.1 and 12.5 μM, respectively, whereas Lys-Pg binds with high affinity to both Fg and Fm (Kd values of 0.25 and 0.21 μM, respectively). In contrast, Mini-Pg demonstrates weak binding to Fg and Fm with Kd values of 10.5 and 24.5 μM, respectively. To complement the binding experiments, kinetic studies of Pg activation by t-PA were performed in the absence or presence of native Fn clots by monitoring Pn formation using a Pn-directed chromogenic substrate. As expected, the catalytic efficiency of Lys-Pg or Mini-Pg activation by t-PA in the absence of cofactor was higher than that of Glu-Pg (kcat/KM values of 1.3, 0.325, and 0.026 μM−1 min−1, respectively). The catalytic efficiency of Glu-Pg activation by t-PA is 500-fold higher in the presence of Fn than it is in its absence. The stimulatory effect of fibrin was maintained with Lys and Mini-Pg with over 100-fold enhancement in catalytic efficiency of activation. The fibrin-dependent increase in catalytic efficiency was expressed predominantly through a decrease in KM, with values of 〉20 and 0.2 μM for Glu-Pg activation in the absence and presence of fibrin, respectively. Lys and Mini-Pg also expressed similar enhancements in catalytic efficiency through a decrease in KM. Thus, despite a 100-fold range in their affinities for Fn, the activation of Mini-Pg, Glu-Pg and Lys-Pg by t-PA are all enhanced by at least 2 orders of magnitude in the presence of Fn. These results demonstrate that substrate binding to Fn is not essential for Fn-mediated stimulation of Pg activation by t-PA. To investigate the importance of the Fn-plasminogen activator interaction, activation studies were carried out using urokinase-type plasminogen activator (u-PA), an activator without Fn affinity. In contrast to the results with t-PA, Fn did not enhance u-PA-mediated activation of Glu, Lys or Mini-Pg. The lack of fibrin stimulation of Pg activation by u-PA suggests that the Pg-Fn interaction is not essential to Pg activation. Therefore, the cofactor role of Fn is expressed predominantly through interaction and stimulation of t-PA. These findings support the hypothesis that Fn binding to t-PA may expose cryptic binding sites in the activator that stabilize the formation of the enzyme-substrate complex.

Beschreibung: Therapeutic plasma exchange is an effective empiric treatment for thrombotic thrombocytopenic purpura (TTP), but how therapy affects the level of adisintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) or inhibitor has not been reported in many patients. We prospectively analyzed ADAMTS13 activity and inhibitor levels in 37 adults with TTP. ADAMTS13 level at presentation was lower than 5% in 16 of 20 patients with idiopathic TTP and in none of 17 patients with TTP associated with hematopoietic stem cell transplantation, cancer, drugs, or pregnancy (P 〈 .00001). Seven of the 16 patients with ADAMTS13 activity lower than 5% (≈ 44%) had inhibitors. For 8 patients followed serially with ADAMTS13 activity lower than 5% but no inhibitor at presentation, plasma exchange led to complete clinical remission and a rise in ADAMTS13 level. In contrast, 4 patients with low ADAMTS13 activity but high-titer inhibitor (〉 5 units/mL) had neither a rise in ADAMTS13 activity nor a reduction in the inhibitor titer: 3 had recurrent disease and 1 died. Among 17 patients with AD-AMTS13 activity at presentation higher than 25%, 10 died. Mortality rate for idiopathic TTP was 15%, whereas mortality for nonidiopathic TTP was 59% (P 〈 .02). We conclude that assays of ADAMTS13 activity and inhibitors in addition to the clinical categories (idiopathic TTP and nonidiopathic TTP) are predictive of outcome and may be useful to tailor patient treatment.

Beschreibung: Neuroblastoma(NB) is a common solid tumors in children. Even with active chemotherapy, operation and irradiation, the survival rate is still very low in advanced patients. To improve the treatment results, autolougous haematopoietic stem cell transplantations have been performed in 11 patients with advanced NB in our hospital. The average age was 3.8 years old (2–6 years old) and the average weight was 15.3kg(11.6–20kg) in this group. All 9 patients were in stage IV with the primary mass in abdomen and extensive bone marrow involvement but 2 patients in stage III with the primary mass in thorax. Although high dose chemotherapy and active operation was given, 4 cases still have not got complete remission at the time of transplant. As 2 cases received 2 times transplant, totally 13 transplants were performed in these 11 children. 3 were collected stem cell from the anterior crista of iliac in both sides while another 10 were received the stem cell from the peripheral mononuclear cell harvested with CS-3000 cell separator after G-CSF mobilization. To reduce the risk of post transplant relapse, 4 cases were purged with CliniMACS based on the CD34 positive selection. All the patients conditioned with Etopside plus Carboplatin plus Melphalan. Results show the number of mononuclear cell collected from bone marrow or peripheral blood was equal to 5.7±0.9′108/kg and 5.7±1.0′108/kg respectively(p〉0.05). All of them achieved the haematopoietic reconstitution after transplantation. The mean time for the neutrophile count recovering to 0.5 ′109/L was 10.5±5.7 days and the platelet recovering over 2.0 ′109/L. The mean transfusion independent day was 16.8±9.4 days with average 2 units of packed red blood cells and 4 units of platelet products transfused in the course of transplantation. The mean fellow up time was 26.7 months. 4/11 children died of relapse 5 months to 27 months after transplantation. Other 7 children are still in survival. None of our children died of complication associated with transplantation. It suggests autologous stem cell transplantation is a safe and effective measure in the treatment of children with advanced neuroblastoma and worth of further recommendation.