The interaction of acute myeloid leukemia (AML) blasts with the leukemic microenvironment is postulated to be an important mediator of resistance to chemotherapy and disease relapse. We hypothesized that inhibition of the CXCR4/CXCL12 axis by the small molecule inhibitor, plerixafor, would disrupt the interaction of leukemic blasts with the environment and increase the sensitivity of AML blasts to chemotherapy. In this phase 1/2 study, 52 patients with relapsed or refractory AML were treated with plerixafor in combination with mitoxantrone, etoposide, and cytarabine. In phase 1, plerixafor was escalated to a maximum of 0.24 mg/kg/d without any dose-limiting toxicities. In phase 2, 46 patients were treated with plerixafor 0.24 mg/kg/d in combination with chemotherapy with an overall complete remission and complete remission with incomplete blood count recovery rate (CR + CRi) of 46%. Correlative studies demonstrated a 2-fold mobilization in leukemic blasts into the peripheral circulation. No evidence of symptomatic hyperleukocytosis or delayed count recovery was observed with the addition of plerixafor. We conclude that the addition of plerixafor to cytotoxic chemotherapy is feasible in AML, and results in encouraging rates of remission with correlative studies demonstrating in vivo evidence of disruption of the CXCR4/CXCL12 axis.

Between 1996 and 1999, 172 patients (median age, 42 years) with hematologic malignancies were randomly assigned to receive either HLA-identical related bone marrow or G-CSF-mobilized peripheral blood mononuclear cells (G-PBMCs) after myeloablative conditioning. Early results showed that transplantation of G-PBMCs, compared with marrow, was associated with significantly superior 2-year disease-free survival (DFS) and overall survival. Ten-year follow-up showed a sustained DFS benefit associated with G-PBMCs (mortality or relapse hazard ratio, 0.64; 95% confidence interval, 0.4-1.0; P = .03), although the likelihood of overall survival was not significantly different between the 2 groups (mortality hazard ratio, 0.75; 95% confidence interval, 0.5-1.2; P = .20). The 10-year cumulative incidence of chronic GVHD and the duration of systemic immunosuppression were similar in the 2 groups. In summary, transplantation of HLA-identical related G-PBMCs, compared with marrow, was associated with superior short-term and long-term DFS, and there was no evidence that this benefit was outweighed by GVHD-related late mortality.

Donor lymphocyte infusion (DLI), a standard relapse treatment after allogeneic stem cell transplantation (AlloSCT), has limited efficacy and often triggers GVHD. We hypothesized that after AlloSCT tumor-infiltrating donor lymphocytes could be costimulated ex vivo to preferentially activate/expand antitumor effectors. We tested the feasibility and safety of costimulated, tumor-derived donor lymphocyte (TDL) infusion in a phase 1 trial. Tumor was resected from 8 patients with B-cell malignancy progression post-AlloSCT; tumor cell suspensions were costimulated with anti-CD3/anti-CD28 Ab-coated magnetic beads and cultured to generate TDL products for each patient. Costimulation yielded increased proportions of T-bet(+)FoxP3(-) type 1 effector donor T cells. A median of 2.04 × 10(7) TDL/kg was infused; TDLs were well tolerated, notably without GVHD. Two transient positron emission tomography (PET) responses and 2 mixed responses were observed in these refractory tumors. TDL are a feasible, tolerable, and novel donor cell therapy alternative for relapse after AlloSCT.

To assess whether antithrombotic prophylaxis with low-molecular-weight heparin effectively prevents recurrence of late pregnancy complications, 135 women with previous history of preeclampsia, hemolytic anemia, elevated liver enzymes and low platelet count syndrome, intrauterine fetal death, fetal growth restriction, or placental abruption who had been referred within the 12th gestational week were randomized to medical surveillance alone (n = 68) or combined to open-label nadroparin (3800 IU daily subcutaneous injections) treatment (n = 67) in the setting of a randomized, parallel-group, superiority trial, run in Italy from April 2007 to April 2010. Primary outcome was a composite end point of late-pregnancy complications. Analysis was by intention to treat. The study was stopped for futility at the time of the first planned interim analysis. Among the 128 women eventually available for final analyses, 13 of the 63 (21%) randomized to nadroparin compared with 12 of the 65 (18%) on medical surveillance alone progressed to the primary end point. The absolute event risk difference between treatment arms (2.2; -1.6 to 16.0) was not statistically significant (P = .76). Thus, nadroparin did not prevent late-pregnancy complications in women at risk of recurrence. This finding challenges the role of antithrombotic prophylaxis with low-molecular-weight heparin in the prevention of recurrent late pregnancy complications The trial was registered at http://ricerca-clinica.agenziafarmaco.it as EudraCT 2006-004205-26.

Candidate genetic associations with acute GVHD (aGVHD) were evaluated with the use of genotyped and imputed single-nucleotide polymorphism data from genome-wide scans of 1298 allogeneic hematopoietic cell transplantation (HCT) donors and recipients. Of 40 previously reported candidate SNPs, 6 were successfully genotyped, and 10 were imputed and passed criteria for analysis. Patient and donor genotypes were assessed for association with grades IIb-IV and III-IV aGVHD, stratified by donor type, in univariate and multivariate allelic, recessive and dominant models. Use of imputed genotypes to replicate previous IL10 associations was validated. Similar to previous publications, the IL6 donor genotype for rs1800795 was associated with a 20%-50% increased risk for grade IIb-IV aGVHD after unrelated HCT in the allelic (adjusted P = .011) and recessive (adjusted P = .0013) models. The donor genotype was associated with a 60% increase in risk for grade III-IV aGVHD after related HCT (adjusted P = .028). Other associations were found for IL2, CTLA4, HPSE, and MTHFR but were inconsistent with original publications. These results illustrate the advantages of using imputed single-nucleotide polymorphism data in genetic analyses and demonstrate the importance of validation in genetic association studies.

The aim of this phase 2 study was to evaluate the efficacy and safety of trastuzumab, a humanized monoclonal antibody targeted against the human epidermal growth factor receptor 2 (HER2), for adult patients with relapsed/refractory HER2-positive B-ALL. Fifteen patients, with a median age of 62 years, received trastuzumab according to the schedule approved for breast cancer patients (ie, 4 mg/kg intravenous loading dose followed by 2 mg/kg weekly). The overall response rate was 13% with 2 patients achieving partial response and partial remission cytolytic response, respectively. Two other patients were documented with blast clearance. Only 1 reversible grade 3 cardiac toxic event occurred. This phase 2 study showed that trastuzumab monotherapy can allow for some responses in a very high-risk refractory/relapsed HER2-positive adult B-ALL population. Combination of trastuzumab with chemotherapy or other therapeutic monoclonal antibodies should be tested in the future.

This prospective study investigated the efficacy, safety, and response duration of low-dose rituximab (100 mg fixed dose for 4 weekly infusions) together with a short course of steroids as first- or second-line therapy in 23 patients with primary autoimmune hemolytic anemia (AIHA). The overall response was 82.6% at month +2, and subsequently stabilized to ∼ 90% at months +6 and +12; the response was better in warm autoimmune hemolytic anemia (WAIHA; overall response, 100% at all time points) than in cold hemagglutinin disease (CHD; average, 60%); the relapse-free survival was 100% for WAIHA at +6 and +12 months versus 89% and 59% in CHD, respectively, and the estimated relapse-free survival at 2 years was 81% and 40% for the warm and cold forms, respectively. The risk of relapse was higher in CHD and in patients with a longer interval between diagnosis and enrollment. Steroid administration was reduced both as cumulative dose (∼ 50%) and duration compared with the patient's past history. Treatment was well tolerated and no adverse events or infections were recorded; retreatment was also effective. The clinical response was correlated with amelioration biologic markers such as cytokine production (IFN-γ, IL-12, TNF-α, and IL-17), suggesting that low-dose rituximab exerts an immunomodulating activity. This study is registered at www.clinicaltrials.gov as NCT01345708.

This was a 24-week, multicenter phase-2 study designed to assess safety, tolerability, and pharmacodynamics of FBS0701, a novel oral chelator, in adults with transfusional iron overload. Fifty-one patients, stratified by transfusional iron intake, were randomized to FBS0701 at either 14.5 or 29 mg/kg/d (16 and 32 mg/kg/d salt form). FBS0701 was generally well tolerated at both doses. Forty-nine patients (96%) completed the study. There were no drug-related serious adverse events. No adverse events (AEs) showed dose-dependency in frequency or severity. Treatment-related nausea, vomiting, abdominal pain, and diarrhea were each noted in < 5% of patients. Mean serum creatinine did not change significantly from Baseline or between dose groups. Transaminases wer increased in 8 (16%), three of whom acquired HCV on-study from a single blood bank while five had an abnormal baseline ALT. The 24 week mean change in liver iron concentration (ΔLIC) at 14.5 mg/kg/d was +3.1 mg/g (dw); 29% achieved a decrease in LIC. Mean ΔLIC at 29 mg/kg/d was -0.3 mg/g (dw); 44% achieved a decrease in LIC (P < .03 for ΔLIC between doses). The safety and tolerability profile at therapeutic doses compare favorably to other oral chelators.

Strategies that augment a GVL effect without increasing the risk of GVHD are required to improve the outcome after allogeneic stem cell transplantation (SCT). Azacitidine (AZA) up-regulates the expression of tumor Ags on leukemic blasts in vitro and expands the numbers of immunomodulatory T regulatory cells (Tregs) in animal models. Reasoning that AZA might selectively augment a GVL effect, we studied the immunologic sequelae of AZA administration after allogeneic SCT. Twenty-seven patients who had undergone a reduced intensity allogeneic transplantation for acute myeloid leukemia were treated with monthly courses of AZA, and CD8(+) T-cell responses to candidate tumor Ags and circulating Tregs were measured. AZA after transplantation was well tolerated, and its administration was associated with a low incidence of GVHD. Administration of AZA increased the number of Tregs within the first 3 months after transplantation compared with a control population (P = .0127). AZA administration also induced a cytotoxic CD8(+) T-cell response to several tumor Ags, including melanoma-associated Ag 1, B melanoma antigen 1, and Wilm tumor Ag 1. These data support the further examination of AZA after transplantation as a mechanism of augmenting a GVL effect without a concomitant increase in GVHD.

Solulin is a soluble form of thrombomodulin that is resistant to proteolysis and oxidation. It has been shown to increase the clot lysis time in factor VIII (fVIII)-deficient plasma by an activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-dependent mechanism. In the present study, blood was drawn from humans and dogs with hemophilia, and thromboelastography was used to measure tissue factor-initiated fibrin formation and tissue-plasminogen activator-induced fibrinolysis. The kinetics of TAFI and protein C activation by the thrombin-Solulin complex were determined to describe the relative extent of anticoagulation and antifibrinolysis. In severe hemophilia A, clot stability increased by > 4-fold in the presence of Solulin while minimally affecting clot lysis time. Patients receiving fVIII/fIX prophylaxis showed a similar trend of increased clot stability in the presence of Solulin. The catalytic efficiencies of TAFI and protein C activation by the thrombin-Solulin complex were determined to be 1.53 and 0.02/μM/s, respectively, explaining its preference for antifibrinolysis over anticoagulation at low concentrations. Finally, hemophilic dogs given Solulin had improved clot strength in thromboelastography assays. In conclusion, the antifibrinolytic properties of Solulin are exhibited in hemophilic human (in vitro) and dog (in vivo/ex vivo) blood at low concentrations. Our findings suggest the therapeutic utility of Solulin at a range of very low doses.

Essential thrombocythemia (ET) is characterized by enhanced platelet generation and thrombotic complications. Once-daily low-dose aspirin incompletely inhibits platelet thromboxane A(2) (TXA(2)) in the majority of ET patients. In the present study, we investigated the determinants of aspirin-insensitive platelet TXA(2) biosynthesis and whether it could be further suppressed by changing the aspirin dose, formulation, or dosing interval. In 41 aspirin-treated ET patients, the immature platelet count predicted serum TXB(2) independently of platelet count, age, JAK-2 V617F mutation, or cytoreduction (β = 3.53, P = .001). Twenty-one aspirin-treated patients with serum TXB(2) ≥ 4 ng/mL at 24 hours after dosing were randomized to the following 7-day regimens in a crossover design: enteric-coated aspirin 100 mg twice daily, enteric-coated aspirin 200 mg once daily, or plain aspirin 100 mg once daily. A twice-daily regimen caused a further 88% median (IQR, 78%-92%, P < .001) TXB(2) reduction and normalized the functional platelet response to aspirin, as assessed by urinary 11-dehydro-TXB(2) excretion and the VerifyNow Aspirin assay. Doubling the aspirin dose reduced serum TXB(2) only partially by 39% median (IQR, 29%-54%, P < .05). We conclude that the abnormal megakaryopoiesis characterizing ET accounts for a shorter-lasting antiplatelet effect of low-dose aspirin through faster renewal of platelet cyclooxygenase-1, and impaired platelet inhibition can be rescued by modulating the aspirin dosing interval rather than the dose.

Iron supplementation strategies in the developing world remain controversial because of fears of exacerbating prevalent infectious diseases. Understanding the conditions in which iron will be absorbed and incorporated into erythrocytes is therefore important. We studied Gambian children with either postmalarial or nonmalarial anemia, who were given oral iron supplements daily for 30 days. Supplements administered on days 1 and 15 contained the stable iron isotopes (57)Fe and (58)Fe, respectively, and erythrocyte incorporation was measured in blood samples drawn 14 days later. We investigated how the iron-regulatory hormone hepcidin and other inflammatory/iron-related indices, all measured on the day of isotope administration, correlated with erythrocyte iron incorporation. In univariate analyses, hepcidin, ferritin, C-reactive protein, and soluble transferrin receptor (sTfR) strongly predicted incorporation of (57)Fe given on day 1, while hepcidin, ferritin, and sTfR/log ferritin correlated with (58)Fe incorporation. In a final multivariate model, the most consistent predictor of erythrocyte isotope incorporation was hepcidin. We conclude that under conditions of competing signals (anemia, iron deficiency, and infection), hepcidin powerfully controls use of dietary iron. We suggest that low-cost point-of-care hepcidin assays would aid iron supplementation programs in the developing world.

Current factor VIII (FVIII) products display a half-life (t(1/2)) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG(1) to extend circulating rFVIII t(1/2). This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t(1/2), 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.

The benefit of adding rituximab to standard treatment in nonsplenectomized patients with primary immune thrombocytopenia (ITP) is uncertain. We performed a pilot randomized trial to determine the feasibility of recruitment, protocol adherence, and blinding of a larger trial of rituximab versus placebo; and to evaluate the potential efficacy of adjuvant rituximab in ITP. Nonsplenectomized adults with newly diagnosed or relapsed ITP who were receiving standard ITP therapy for a platelet count below 30 × 10(9)/L were randomly allocated to receive 4 weekly infusions of 375 mg/m(2) rituximab or saline placebo. Sixty patients were recruited over 46 months, which was slower than anticipated. Protocol adherence and follow-up targets were achieved, and blinding was successful for research staff but not for patients. After 6 months, there was no difference between rituximab and placebo groups for the composite outcome of any platelet count below 50 × 10(9)/L, significant bleeding or rescue treatment once standard treatment was stopped (21/32 [65.6%] vs 21/26 [80.8%]; relative risk = 0.81, 95% confidence intervals, 0.59%-1.11%). Timely accrual poses a challenge to the conduct of a large randomized trial of rituximab for presplenectomy ITP. No difference in the frequency of the composite outcome was observed in this pilot trial (registered at www.clinicaltrials.gov NCT00372892).

We previously reported that remission duration < 1 year, extranodal disease, and B symptoms before salvage chemotherapy (SLT) can stratify relapsed or refractory Hodgkin lymphoma (HL) patients into favorable and unfavorable cohorts. In addition, pre-autologous stem cell transplant (ASCT) (18)FDG-PET response to SLT predicts outcome. This phase 2 study uses both pre-SLT prognostic factors and post-SLT FDG-PET response in a risk-adapted approach to improve PFS after high-dose radio-chemotherapy (HDT) and ASCT. The first SLT uses 2 cycles of ICE in a standard or augmented dose (ICE/aICE), followed by restaging FDG-PET scan. Patients with a negative scan received a transplant. If the FDG-PET scan remained positive, patients received 4 biweekly doses of gemcitabine, vinorelbine, and liposomal doxorubicin. Patients without evidence of disease progression proceeded to HDT/ASCT; those with progressive disease were study failures. At a median follow-up of 51 months, EFS analyzed by intent to treat as well as for transplanted patients is 70% and 79%, respectively. Patients transplanted with negative FDG-PET, pre-HDT/ASCT after 1 or 2 SLT programs, had an EFS of > 80%, versus 28.6% for patients with a positive scan (P < .001). This prospective study provides evidence that the goal of SLT in patients with Hodgkin lymphoma should be a negative FDG-PET scan before HDT/ASCT.

Adenosine acts as anti-inflammatory mediator on the immune system and has been described in regulatory T cell (Treg)-mediated suppression. In the absence of adenosine deaminase (ADA), adenosine and other purine metabolites accumulate, leading to severe immunodeficiency with recurrent infections (ADA-SCID). Particularly ADA-deficient patients with late-onset forms and after enzyme replacement therapy (PEG-ADA) are known to manifest immune dysregulation. Herein we provide evidence that alterations in the purine metabolism interfere with Treg function, thereby contributing to autoimmune manifestations in ADA deficiency. Tregs isolated from PEG-ADA-treated patients are reduced in number and show decreased suppressive activity, whereas they are corrected after gene therapy. Untreated murine ADA(-/-) Tregs show alterations in the plasma membrane CD39/CD73 ectonucleotidase machinery and limited suppressive activity via extracellular adenosine. PEG-ADA-treated mice developed multiple autoantibodies and hypothyroidism in contrast to mice treated with bone marrow transplantation or gene therapy. Tregs isolated from PEG-ADA-treated mice lacked suppressive activity, suggesting that this treatment interferes with Treg functionality. The alterations in the CD39/CD73 adenosinergic machinery and loss of function in ADA-deficient Tregs provide new insights into a predisposition to autoimmunity and the underlying mechanisms causing defective peripheral tolerance in ADA-SCID.

This multicenter, first-in-human study evaluated the safety, tolerability, and pharmacokinetic and pharmacodynamic properties of the anti-CS1 monoclonal antibody elotuzumab. A standard 3 + 3 design was used to determine maximum tolerated dose; dose-limiting toxicities were assessed during cycle 1. Thirty-five patients with relapsed/refractory multiple myeloma were treated with intravenous elotuzumab at doses ranging from 0.5 to 20 mg/kg every 2 weeks. Patients who achieved at least stable disease after 4 treatments could receive another 4 treatments. No maximum tolerated dose was identified up to the maximum planned dose of 20 mg/kg. The most common adverse events, regardless of attribution, were cough, headache, back pain, fever, and chills. Adverse events were generally mild to moderate in severity, and adverse events attributed to study medication were primarily infusion-related. Plasma elotuzumab levels and terminal half-life increased with dose whereas clearance decreased, suggesting target-mediated clearance. CS1 on bone marrow-derived plasma cells was reliably saturated (≥ 95%) at the 10-mg/kg and 20-mg/kg dose levels. Using the European Group for Bone and Marrow Transplantation myeloma response criteria, 9 patients (26.5%) had stable disease. In summary, elotuzumab was generally well tolerated in this population, justifying further exploration of this agent in combination regimens.

Although below-knee compression elastic stockings (CES) are effective for the prevention of the postthrombotic syndrome (PTS), a substantial number of patients with deep venous thrombosis still develop PTS. In the present open-label, randomized clinical trial, we compared thigh-length with below-knee CES for the prevention of PTS. A total of 267 patients with the first episode of proximal deep venous thrombosis were randomized to wear either thigh-length or below-knee CES for 2 years. After 3, 6, 12, 18, 24, and 36 months, they were assessed for PTS manifestations according to the Villalta scale. PTS developed in 44 (32.6%) of the 135 patients randomized to thigh-length CES and in 47 (35.6%) of the 132 allocated to below-knee CES, for an adjusted hazard ratio of 0.93 (95% confidence interval, 0.62-1.41). Severe PTS developed in 3 patients in each group. CES-related side effects developed in 55 (40.7%) of the 135 patients allocated to thigh-length CES and in 36 (27.3%) of those randomized to the below-knee group (P = .017), and led to premature discontinuation of their use in 29 (21.5%) and 18 (13.6%) patients, respectively. We conclude that thigh-length CES do not offer a better protection against PTS than below-knee CES and are less well tolerated.

Dasatinib is a highly potent BCR-ABL inhibitor with established efficacy and safety in imatinib-resistant/-intolerant patients with chronic myeloid leukemia (CML). In the phase 3 DASISION trial, patients with newly diagnosed chronic-phase (CP) CML were randomized to receive dasatinib 100 mg (n = 259) or imatinib 400 mg (n = 260) once daily. Primary data showed superior efficacy for dasatinib compared with imatinib after 12 months, including significantly higher rates of complete cytogenetic response (CCyR), confirmed CCyR (primary end point), and major molecular response (MMR). Here, 24-month data are presented. Cumulative response rates by 24 months in dasatinib and imatinib arms were: CCyR in 86% versus 82%, MMR in 64% versus 46%, and BCR-ABL reduction to ≤ 0.0032% (4.5-log reduction) in 17% versus 8%. Transformation to accelerated-/ blast-phase CML on study occurred in 2.3% with dasatinib versus 5.0% with imatinib. BCR-ABL mutations, assessed after discontinuation, were detected in 10 patients in each arm. In safety analyses, fluid retention, superficial edema, myalgia, vomiting, and rash were less frequent with dasatinib compared with imatinib, whereas pleural effusion and grade 3/4 thrombocytopenia were more frequent with dasatinib. Overall, dasatinib continues to show faster and deeper responses compared with imatinib, supporting first-line use of dasatinib in patients with newly diagnosed CML-CP. This study was registered at ClinicalTrials.gov: NCT00481247.

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.