ALBERT

Description: Red cell and platelet transfusion requirements have been reported to be lower following reduced intensity conditioning allogeneic hematopoietic stem cell transplantation (RIC allo-HCT) when compared to myeloablative (MA) allo-HCT. However, previous studies examined RIC regimens with lower haemopoietic toxicity than many of the regimens in use today. We investigated risk factors for red cell and platelet transfusion in patients enrolled prospectively in an Australian study investigating the non-HLA immunogenetics of sibling allo-HCT. The transfusion requirements in the first year post transplant were reviewed for 122 patients transplanted between 2002 and 2006 in three Australian transplant centres. Seventy-one patients received MA and 51 RIC regimens. Using regression analysis, the outcome variables of total red cell and platelet units transfused were analysed. The factors age, transplant centre, disease, transplant type (RIC v MA), days of neutropenia, death within 12 months, disease risk (high risk (HR) v standard risk (SR)) and ABO mismatch underwent univariate analysis. Associated variables with p

Description: HCD122 is a novel, fully human, IgG1 antagonistic monoclonal antibody targeting the CD40 receptor. This antibody blocks CD40-mediated signaling and is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Previous preclinical investigation confirmed expression of CD40 on myeloma cells in the majority of patients and reported antitumor activity of HCD122 against multiple myeloma cells ex vivo (Tai, Y et al. Cancer Res2005; 65(13): 5898–5906). This ongoing phase 1 study will determine the maximum tolerated dose of CHIR-12.12 in multiple myeloma patients (pts) who are relapsed or refractory after at least one prior therapy. Planned dose levels are 1, 3 and 10 mg/kg administered IV once weekly for 4 weeks. Each dose group will enroll 3–6 pts to evaluate safety, pharmacokinetics (PK) and clinical response. To date, 9 pts have been treated at 2 dose levels: 3 pts at 1 mg/kg and 6 pts at 3 mg/kg. Median patient age is 65 yrs (46–81 yrs); median number of prior therapies is 3 (2–12). No dose limiting toxicity (DLT) occurred at the 1mg/kg dose level. At 3 mg/kg, 1 DLT of grade 4 thrombocytopenia occurred in 1 pt. No other grade 3 and 4 lab abnormalities and adverse events have been reported. In 7 pts with available data, infusions were well tolerated, with easily managed grade 1–2 toxicities, primarily chills (5 pts), nausea (3 pts), pyrexia (2 pts), and arthralgia (2 pts) mainly reported during the first infusion. Preliminary PK analysis showed more than dose proportional - increase in Cmax and AUC at the 3 mg/kg dose level compared to the 1 mg/kg dose level. At the 3 mg/kg dose, antibody accumulation occurred week-to-week; the mean Cmax after the fourth infusion on Day 22 was 126.1 mg/mL(range 52 – 195 ug/mL) and HCD122 levels were measurable up to Day 57 and in one patient up to Day 99. One week after the last 3 mg/kg dose, trough levels ranged from 28 to 109 mg/mL. Of the 3 pts at 1 mg/kg, one showed stable disease (SD) for 〉23 weeks and two had progressive disease (PD) by week 5. Of the 6 pts at 3 mg/kg, one had partial response (PR) at week 9 and was confirmed at week 15, one had SD for 〉 5 weeks, and 4 had PD at week 5. One pt with PD terminated the study before final safety evaluation, and must be replaced before assessment of the 3mg/kg dose level is complete. Thus, in preliminary studies, HCD122 appears to be safe and well tolerated to date at doses of 1 mg/kg and 3 mg/kg weekly for 4 doses and shows promising anti-myeloma activity. Enrollment is continuing to determine MTD.

Description: Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

Description: Increasing the expression of the gamma globin genes is considered a useful therapeutic approach to the beta globin diseases. Because butyrate and alpha-amino-n-butyric acid (ABA) augment gamma globin expression in normal neonatal and adult erythroid progenitors, we investigated the effects of sodium butyrate and ABA on erythroid progenitors of patients with beta thalassemia and sickle cell anemia who might benefit from such an effect. Both substances increased fetal hemoglobin (Hb F) expression in Bfu-e from 7% to 30% above levels found in control cultures from the same subjects with sickle cell anemia. The fraction of cultured erythroblasts producing Hb F increased more than 20% with sodium butyrate treatment in 70% of cultures. In most cultures, this produced greater than 20% total Hb F and greater than 70% F cells, levels which have been considered beneficial in ameliorating clinical symptoms. Alpha: non-alpha (alpha-non-alpha) imbalance was decreased by 36% in erythroid progenitors of patients with beta thalassemia cultured in the presence of butyrate compared with control cultures from the same subjects. These data suggest that sodium butyrate may have therapeutic potential for increasing gamma globin expression in the beta globin diseases.

Description: The previous studies assessing the relationship of cytogenetic abnormalities to JAK2 status have not shown any significant associations, although numbers have generally been too small to make a definitive comment. We therefore assessed the JAK2 V617F mutation status of 337 patients from 39 centers (175 males, 161 females; median age at time of study 71 years old, range 32–98). Patients were classified as follows: 100 with typical MDS, 10 with 5q- syndrome, 17 with AREB-2, 2 with RARS, 13 with RARS-T, 23 with MPD/MDS, 12 with CMML, 142 with typical MPDs, 5 with hypereosinophilic syndrome (HES), 7 with AML with multi-lineage dysplasia from Ph- MPD, 10 with aCML Ph- or MPD/MDS-u and 25 with typical CML Ph+. Bone marrow or blood derived genomic DNA was screened for the JAK2 mutation. The JAK2 V617F mutation was found in 109/337 pts (32%): typical MPD-Ph− (47%) cases, MDS (4.5%), 5q- syndrome (60%), AREB-2 (64%), CMML (42%), MDS/MPD (36%). A clonal cytogenetic abnormality was detected by R-banding in 159/337 cases (47%). The JAK2 mutation was associated with a chromosomal abnormality in 81/159 cases (51%). The most common chromosome abnormality associated with JAK2 mutation was the gain of chromosome 9 (n= 22, Associated with JAK2 mutation: 19/22 cases 86%). The second most common abnormality was partial deletion of 20q (n=30, Associated with JAK2 mutation: 19/30 cases 63%). The partial or complete loss of chromosome 7 (n=16, Associated with JAK2 mutation: 6/11), deletion of 5q- (n=10, Associated with JAK2 mutation: 6/10), gain of chromosome 8 (n=11, Associated with JAK2 mutation: 7/11), partial deletion of 11q (n=8, Associated with JAK2 mutation: 5/8), partial deletion of 12p (n=6 Associated with JAK2 mutation 4/6), partial deletion of 13q (n=5, Associated with JAK2 mutation: 4/5). Patients with trisomy 9 and JAK2 mutation were classified as follows : polycytemia vera (PV) n=4 (100% JAK2 mutated), essential thrombocytemia (ET) n=3 (100% JAK2 mutated), idiopathic myelofibrosis (IMF), n=2 (100% JAK2 mutated), MPD/MDS, n=3 (67% JAK2 mutated), secondary AML post MDS, n=1 (JAK2 mutated), atypical MPD, n=6 (100% JAK2 mutated), MMCL, n=1(JAK2 mutated). The V617 mutation was not found in the case of de novo AML, and 2 typical MDS cases. 9 males, 10 females, the median age: 68 years old, range 40–98 years old, median white cell count (WCC) 11 G/l, range 2.4–50.7 G/l (13/19 pts〉 10G/l), median hemoglobin 13.5 g/dl, range 9–20.2 g/dl (5/19 pts〉 17g/dl), median platelet count 667G/l range 9–1769 G/l (9/19 pts〉 500 G/l). The BCR/ABL transcript (multiplex PCR) was not detected in all of these cases. In this study the gain of chromosome 9 associated with JAK2 V617F mutation was the most frequent chromosome abnormality (100%) observed in typical MPDs and atypical syndrome such as MDS/MPD. In summary, previous studies assessing the relationship of cytogenetic abnormalies to JAK2 status did not show any significant association (ref). The del(20q), del(13q), trisomy 8 and del(5q) are known to be recurring non-specific cytogenetic abnormalities, and some of them are also detectable in patients with JAK2 mutation positive or negative. We describe here a significant association the JAK2 V617F mutation and trisomy of chromosome 9 that was detected in a cohort of patients with gain of chromosome 9. Clearly, in addition to PV, IMF, and ET these associations were found in other disease entities, high frequency in the case of atypical MPDs particularly in the case of aCML. Previously, Campbell et al. reported 10 patients with a trisomy 9, in typical MPDs, all of them were V617F+. A longer follow-up and morphological diagnosis is however necessary to determine the prognostic signification of JAK2 mutation and +9 in the cases of classic myeloproliferative syndromes and atypical syndrome such as MDS/MPD overlap syndrome.

Description: Despite the established contribution of platelets to thrombotic cardiovascular disorders, documented in part by the effectiveness of platelet function inhibitors and the increased risk of thrombosis associated with high normal and supranormal platelet counts, relationships between circulating platelet count and thrombotic events remain largely undefined. Since the initiation and propagation of arterial, platelet-dependent thrombus must depend upon platelet count, albeit in a manner that could be nonlinear, we hypothesized that reducing platelet count within the normal range would produce an anti-thrombotic benefit with minimal effects on hemostasis. To test this hypothesis, we reduced the platelet count in baboons (n=4) by targeting the megakaryocyte growth and development factor thrombopoietin (TPO). A polyclonal anti-TPO autoantibody (anti-TPOab) was purified from the serum of a baboon that developed thrombocytopenia following recombinant TPO injections. The IC50 of the purified IgG fraction was found to be 0.76 μg/ml, determined using a proliferation assay with a TPO-dependent cell line. An i.v. bolus of the anti-TPO antiserum, 30–35 ml infused into baboons, resulted in a transient, 〉60% decrease in the circulating platelet count after 2–3 weeks. Other blood cell counts were unaffected vs. baseline values. The effect of platelet count reduction on thrombogenesis was evaluated using an established baboon arteriovenous (AV) shunt thrombosis model. Accumulation of 111-Indium-labeled platelets and 125-Iodine-labeled fibrinogen were measured within a 4 mm i.d. thrombogenic vascular graft segment that was deployed into a chronic AV shunt for 60 min. Blood flow was maintained at 100 ml/min, producing an arterial wall shear rate of 265 sec−1. Standard template bleeding times (BTs) were used to assess hemostatic impairment at various platelet counts. Platelet count reductions, ranging from 46–61% (normal levels averaging 352,000 ± 61,000 platelets/μl), reduced platelet deposition onto the graft surface by 46–68% (vs. control values of 4.1 ± 0.9 x 109 platelets deposited, n=9). Similarly, thrombus fibrin accumulation was reduced by 14–39% (vs. control values of 2.2 ± 0.4 mgs of deposited fibrin). Thrombus formation was not affected acutely by anti-TPOab administration, but correlated directly with circulating platelet numbers. As expected, BTs were not significantly prolonged until platelet counts fell below ~100,000 cells/μl. In contrast, single dose aspirin (32 mg/kg) at normal platelet counts did not significantly reduce graft associated platelet deposition in this model but doubled the BTs to 6.8 ± 2.6 min (vs. control values of 3.4 ± 0.9 min). With further reduction in platelet counts to 90,000 ± 30,000 platelets/μL, BTs were only slightly prolonged (5.6 ± 1.7 min, n=5). When platelet counts averaged 74,000 ± 20,000 platelets/μl in animals given ASA, BTs averaged only 9.4 ± 2.7 min (n=5). Thus ASA produced a hemostatic impairment that was approximately fixed (i.e., a BT prolongation of 3–4 min) and not disproportionately prolonged at reduced platelet counts. Thus specific lowering of the platelet count by pharmacologic inhibition of megakaryocytopoiesis may be an effective anti-thrombotic strategy in populations currently treated with conventional anti-platelet agents. Since direct inhibitors of platelet function produce a significant risk of bleeding, inhibition of platelet production may represent a safer approach for reducing the pro-thrombotic capacity of the circulating platelet pool.

Description: The CD40 antigen is expressed in most B-cell malignancies, including chronic lymphocytic leukemia (CLL), and represents an attractive target for antibody therapy. HCD122 is a high affinity, fully human IgG1 antagonistic anti-CD40 monoclonal antibody designed to exert antitumor activity as an inhibitor of CD40-ligand-mediated survival signals and as a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). In this phase 1 study to determine maximum tolerated dose (MTD) in CLL, eligible patients (pts) include those with relapsed or refractory disease after prior fludarabine treatment and are assigned to receive one cycle of 4 weekly infusions of HCD122 at doses ranging from 0.3 to 10 mg/kg depending upon the dose cohort. A standard phase I design is used with each dose cohort enrolling 3–6 pts for evaluation of MTD, toxicity, pharmacokinetics (PK), and pharmacodynamics (PD). To date, 14 pts have been treated at 3 dose levels: 3 pts at 0.3 mg/kg, 4 pts at 1 mg/kg, and 7 pts at 3 mg/kg. Median patient age was 65 yrs (41–74 yrs); median number of prior therapies was 3 (2–11); median WBC at enrollment was 38.8 K/μ L (3.4–284 K/μ L). No dose limiting toxicity (DLT) occurred at the 0.3 and 1 mg/kg dose levels. At 3 mg/kg, streptococcal sepsis was reported in 1 pt after 2 study infusions and was considered DLT. Transient asymptomatic grade 3 or 4 elevation of amylase and/or lipase occurred in 2 subjects. In 9 pts with available data, infusions were associated with manageable grade 1–2 toxicity, primarily chills (7 pts), nausea (4 pts), and fever (3 pts) that was most predominant with the first infusion. PK analysis showed rapid clearance of HCD122 at the 0.3 and 1mg/kg dose levels, with no detectable levels at 2 and 7 days after infusion, respectively. PK data were available from 6 of 7 patients receiving 3mg/kg. These data indicated that some accumulation of HCD122 occurred at this dose level. Complete antigen saturation data were available for 2 of these subjects. Both showed sustained 100% saturation of antigen on peripheral CD5+CD19+ cells throughout the treatment period. Peripheral CD40+ CLL cells dropped transiently with a mean of 31% during each infusion in the majority of patients but the decreases were not maintained week-to-week. There were no substantive changes in NK or T-cell populations during therapy. In summary, HCD122 was safe and well tolerated up to the 3mg/kg dose level that is currently under evaluation. PK analysis to date suggests that levels higher than 3 mg/kg will be necessary to sustain levels of HCD122 in the expected therapeutic range, and dose escalation will continue to the maximum tolerated dose.

Description: High dose human or mouse activated protein C (APC) treatment is neuroprotective in the transient intraluminal middle cerebral artery occlusion (MCAO) and reperfusion model of ischemic stroke, in mice. However, the therapeutic potential of systemic APC treatment in stroke might be limited because it can disable hemostasis. Low dose thrombomodulin-dependent protein C activator (PCA) enzyme treatment can generate antithrombotic APC on the intraluminal surface without profound systemic anticoagulation and antihemostatic effects in primates. We thus explored whether PCA treatment could produce short term outcome benefits in comparison to fibrinolysis with tPA in the mouse stroke model. Stroke was induced by MCAO between 0 and 60 min using an intraluminal filament that causes progressive and occlusive secondary thrombosis in the MCA region. Cortical perfusion as a measure of thrombotic occlusion was monitored from start until 75 min, i.e., following removal of the intraluminal filament. Cerebral infarct size (% brain volume, TTC stain), edema (% brain volume; calculated), and neurological damage scores (scale 0 – 5) were assessed in survivors at sacrifice next day. All animals (N=12 to 16 in each treatment group) were evaluated for macroscopic hemorrhage (intracerebral or other sites) on autopsy after death. Treatments with vehicle (saline bolus), recombinant PCA thrombin analog W215A/E217A (0.025 mg/kg IV bolus) or plasminogen activator (2.5 mg/kg Bolus, [tPA-B], or 10 mg/kg Infusion for 45 min, [tPA-I]) were started 15 min after initiation of MCAO. One day mortality rates were similar: 29% in the PCA, 38% in the tPA-B, and 23% in the tPA-I treatment groups vs. 23% in vehicle controls (P〉0.05 each). Cerebral infarct size was reduced by 62%, 43%, and 73% in the PCA bolus, tPA-B, and tPA-I treated groups, respectively (p

Description: The protease thrombin is required for normal hemostasis and pathologic thrombogenesis. Since the mechanism of coagulation factor XI (FXI)–dependent thrombus growth remains unclear, we investigated the contribution of FXI to thrombus formation in a primate thrombosis model. Pretreatment of baboons with a novel anti–human FXI monoclonal antibody (aXIMab; 2 mg/kg) inhibited plasma FXI by at least 99% for 10 days, and suppressed thrombin-antithrombin (TAT) complex and β-thromboglobulin (βTG) formation measured immediately downstream from thrombi forming within collagen-coated vascular grafts. FXI inhibition with aXIMab limited platelet and fibrin deposition in 4-mm diameter grafts without an apparent increase in D-dimer release from thrombi, and prevented the occlusion of 2-mm diameter grafts without affecting template bleeding times. In comparison, pretreatment with aspirin (32 mg/kg) prolonged bleeding times but failed to prevent graft occlusion, supporting the concept that FXI blockade may offer therapeutic advantages over other antithrombotic agents in terms of bleeding complications. In whole blood, aXIMab prevented fibrin formation in a collagen-coated flow chamber, independent of factor XII and factor VII. These data suggest that endogenous FXI contributes to arterial thrombus propagation through a striking amplification of thrombin generation at the thrombus luminal surface.