Randomized comparison data on the efficacy and safety of deferasirox for myocardial iron removal in transfusion dependent patients are lacking. CORDELIA was a prospective, randomized comparison of deferasirox (target dose 40 mg/kg per day) vs subcutaneous deferoxamine (50-60 mg/kg per day for 5-7 days/week) for myocardial iron removal in 197 β-thalassemia major patients with myocardial siderosis (T2* 6-20 milliseconds) and no signs of cardiac dysfunction (mean age, 19.8 years). Primary objective was to demonstrate noninferiority of deferasirox for myocardial iron removal, assessed by changes in myocardial T2* after 1 year using a per-protocol analysis. Geometric mean (Gmean) myocardial T2* improved with deferasirox from 11.2 milliseconds at baseline to 12.6 milliseconds at 1 year (Gmeans ratio, 1.12) and with deferoxamine (11.6 milliseconds to 12.3 milliseconds; Gmeans ratio, 1.07). The between-arm Gmeans ratio was 1.056 (95% confidence interval [CI], 0.998, 1.133). The lower 95% CI boundary was greater than the prespecified margin of 0.9, establishing noninferiority of deferasirox vs deferoxamine (P = .057 for superiority of deferasirox). Left ventricular ejection fraction remained stable in both arms. Frequency of drug-related adverse events was comparable between deferasirox (35.4%) and deferoxamine (30.8%). CORDELIA demonstrated the noninferiority of deferasirox compared with deferoxamine for myocardial iron removal. This trial is registered at www.clinicaltrials.gov as #NCT00600938.
For many years there has been considerable disassociation between the understood biology of chronic lymphocytic leukemia (CLL) and the therapeutics used to treat this disease. With the introduction of the first targeted CD20 antibody rituximab and its addition to chemotherapy came the first observation that minimal residual disease–negative (MRD-negative) complete responses (CRs) could be obtained with dramatically improved progression-free survival and overall survival. This advance was soon to be surpassed by the introduction of therapeutics that target B-cell receptor (BCR) signaling. New data show that BCR-inhibiting agents are very active for the treatment of relapsed CLL, despite the lack of MRD-negative CR, with durability of response being considerably more impressive than previously observed with other agents not producing MRD-negative CRs. This perspective provides a view of where these agents may take us in the future as CLL therapy evolves with this exciting new class of drugs.
In this prospective, multicenter, phase 2 study, 64 patients with relapsed or relapsed and refractory multiple myeloma (MM) received up to 8 21-day cycles of bortezomib 1.0 mg/m2 (days 1, 4, 8, and 11), lenalidomide 15 mg/day (days 1-14), and dexamethasone 40/20 mg/day (cycles 1-4) and 20/10 mg/day (cycles 5-8) (days of/after bortezomib dosing). Responding patients could receive maintenance therapy. Median age was 65 years; 66% were male, 58% had relapsed and 42% had relapsed and refractory MM, and 53%, 75%, and 6% had received prior bortezomib, thalidomide, and lenalidomide, respectively. Forty-eight of 64 patients (75%; 90% confidence interval, 65-84) were alive without progressive disease at 6 months (primary end point). The rate of partial response or better was 64%; median duration of response was 8.7 months. Median progression-free and overall survivals were 9.5 and 30 months, respectively (median follow-up: 44 months). Common treatment-related toxicities included sensory neuropathy (53%), fatigue (50%), and neutropenia (42%); common grade 3/4 treatment-related toxicities included neutropenia (30%), thrombocytopenia (22%), and lymphopenia (11%). Grade 3 motor neuropathy was reported in 2 patients. Lenalidomide-bortezomib-dexamethasone appears effective and tolerable in patients with relapsed or relapsed and refractory MM, demonstrating substantial activity among patients with diverse prior therapies and adverse prognostic characteristics. This trial is registered with www.clinicaltrials.gov as #NCT00378209.
The outcome of high-risk (HR) acute lymphoblastic leukemia patients enrolled in the AIEOP-BFM ALL 2000 study in Italy is described. HR criteria were minimal residual disease (MRD) levels ≥10–3 at day 78 (MRD-HR), no complete remission (CR) at day 33, t(4;11) translocation, and prednisone poor response (PPR). Treatment (2 years) included protocol I, 3 polychemotherapy blocks, delayed intensification (protocol IIx2 or IIIx3), cranial radiotherapy, and maintenance. A total of 312 HR patients had a 5-year event-free survival (EFS) of 58.9% (standard error [SE] = 2.8) and an overall survival of 68.9% (SE = 2.6). In hierarchical order, EFS was 45.9% (4.4) in 132 MRD-HR patients, 41.2% (11.9) in 17 patients with no CR at day 33, 36.4% (14.5) in 11 patients with t(4;11), and 74.0% (3.6) in 152 HR patients only for PPR. No statistically significant difference was found for disease-free survival in patients with very HR features [MRD-HR, no CR at day 33, t(4;11) translocation], given hematopoietic stem cell transplantation (HSCT) (n = 66) or chemotherapy only (n = 88), after adjusting for waiting time to HSCT (5.7 months). Patients at HR only for PPR have a favorable outcome. MRD-HR is associated with poor outcome despite intensive treatment and/or HSCT and may qualify for innovative therapies. The study was registered at www.clinicaltrials.gov as #NCT00613457.
Using laser microdissection and mass spectrometry (MS)-based proteomics, we subtyped amyloid deposits from 130 cases of hepatic amyloidosis. Although we confirmed that immunoglobulin light chain amyloidosis was the most frequent cause of hepatic amyloidosis, leukocyte cell-derived chemotaxin 2 (LECT2) amyloidosis (ALect2) accounted for 25% of cases. This novel finding was associated with Hispanic ancestry, incidental discovery of amyloid in liver specimens sampled for other unrelated conditions, and a characteristic pattern of hepatic amyloid deposition. Although ALect2 patients had a common LECT2 polymorphism, pathogenic mutations were not discovered, suggesting that constitutive or compensatory LECT2 overexpression led to ALect2 deposition. These findings indicate that ALect2 is a common cause of hepatic amyloidosis in the population of the United States, and subtyping hepatic amyloid deposits by an accurate analytic method such as MS is required for optimal clinical management of hepatic amyloidosis patients and to avoid incorrect and unnecessarily toxic therapies.
We conducted a pilot trial to investigate the safety and effectiveness of mobilizing CD34+ hematopoietic progenitor cells (HPCs) in adults with β-thalassemia major. We further assessed whether thalassemia patient CD34+ HPCs could be transduced with a globin lentiviral vector under clinical conditions at levels sufficient for therapeutic implementation. All patients tolerated granulocyte colony-stimulating factor well with minimal side effects. All cell collections exceeded 8 x 106 CD34+ cells/kg. Using clinical grade TNS9.3.55 vector, we demonstrated globin gene transfer averaging 0.53 in 3 validation runs performed under current good manufacturing practice conditions. Normalized to vector copy, the vector-encoded β-chain was expressed at a level approximating normal hemizygous protein output. Importantly, stable vector copy number (0.2-0.6) and undiminished vector expression were obtained in NSG mice 6 months posttransplant. Thus, we validated a safe and effective procedure for β-globin gene transfer in thalassemia patient CD34+ HPCs, which we will implement in the first US trial in patients with severe inherited globin disorders. This trial is registered at www.clinicaltrials.gov as #NCT01639690.
Follicular lymphoma (FL) constitutes the second most common non-Hodgkin lymphoma in the western world. FL carries characteristic recurrent structural genomic aberrations. However, information regarding the coding genome in FL is still evolving. Here, we describe the results of massively parallel exome sequencing and single nucleotide polymorphism 6.0 array genomic profiling of 11 highly purified FL cases, and 1 transformed FL case and the validation of selected mutations in 102 FL cases. We report the identification of 15 novel recurrently mutated genes in FL. These include frequent mutations in the linker histone genes HIST1H1 B-E (27%) and mutations in OCT2 (also known as POU2F2; 8%), IRF8 (6%), and ARID1A (11%). A subset of the mutations in HIST1H1 B-E affected binding to DNMT3B, and mutations in HIST1H1 B-E and in EZH2 or ARID1A were largely mutually exclusive, implicating HIST1H1 B-E in epigenetic deregulation in FL. Mutations in OCT2 (POU2F2) affected its transcriptional and functional properties as measured through luciferase assays, the biological analysis of stably transduced cell lines, and global expression profiling. Finally, multiple novel mutated genes located within regions of acquired uniparental disomy in FL are identified. In aggregate, these data substantially broaden our understanding of the genomic pathogenesis of FL.
In this study, we show that microRNA-150 (miR-150) is significantly downregulated in advanced cutaneous T-cell lymphoma (CTCL), and that this downregulation is strongly associated with tumor invasion/metastasis. Inoculation of CTCL cell lines into nonobese diabetic/Shi-scid interleukin 2 (IL-2) null mice led to CTCL cell migration to multiple organs; however, prior transfection of the cells with miR-150 substantially reduced the invasion/metastasis by directly downregulating CCR6, a specific receptor for the chemokine CCL20. We also found that IL-22 and its specific receptor subunit, IL22RA1, were aberrantly overexpressed in advanced CTCL, and that production of IL-22 and CCL20 was increased in cultured CTCL cells. IL22RA1 knockdown specifically reduced CCL20 production in CTCL cells, suggesting that IL-22 upregulation may activate the production of CCL20 and its binding to CCR6, thereby enhancing the multidirectional migration potential of CTCL cells. CTCL cells also exhibited nutrition- and CCL20-dependent chemotaxis, which were inhibited by miR-150 transfection or CCR6 knockdown. From these findings, we conclude that, in the presence of continuous CCR6 upregulation accompanied by miR-150 downregulation, IL-22 activation leads to continuous CCL20-CCR6 interaction in CTCL cells and, in turn, autocrine metastasis to distal organs. This suggests miR-150, CCL20, and CCR6 could be key targets for the treatment of advanced CTCL.
Chronic hepatitis C virus (HCV) infection has been implicated in the induction and maintenance of B-cell lymphomas. The strongest evidence for this derives from clinical observations of tumor regressions upon antiviral treatments. Here we used multiple methods to test the hypothesis that the expansion of HCV-specific B cells gives rise to lymphomas. We obtained lymphoma tissues from HCV-infected lymphoma patients, including some that later regressed upon antiviral treatments. We expressed the lymphoma B-cell receptors as soluble immunoglobulin Gs and membrane IgMs, and analyzed their reactivity with HCV proteins and with HCV virions. We confirmed previous reports that HCV-associated lymphomas use a restricted immunoglobulin variable region gene repertoire. However, we found no evidence for their binding to the HCV antigens. We conclude that most lymphomas of HCV-infected patients do not arise from B cells aimed at eliminating the virus.
Development of resistance to kinase inhibitors remains a clinical challenge. Kinase domain mutations are a common mechanism of resistance in chronic myeloid leukemia (CML), yet the mechanism of resistance in the absence of mutations remains unclear. We tested proteins from the bone marrow microenvironment and found that FGF2 promotes resistance to imatinib in vitro. Fibroblast growth factor 2 (FGF2) was uniquely capable of promoting growth in both short- and long-term assays through the FGF receptor 3/RAS/c-RAF/mitogen-activated protein kinase pathway. Resistance could be overcome with ponatinib, a multikinase inhibitor that targets BCR-ABL and FGF receptor. Clinically, we identified CML patients without kinase domain mutations who were resistant to multiple ABL kinase inhibitors and responded to ponatinib treatment. In comparison to CML patients with kinase domain mutations, these patients had increased FGF2 in their bone marrow when analyzed by immunohistochemistry. Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGF receptor inhibition. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance.
More than 35% of acute myeloid leukemia (AML) patients harbor a constitutively activating mutation in FMS-like tyrosine kinase-3 (FLT3). The most common type, internal tandem duplication (ITD), confers poor prognosis. We report for the first time on TTT-3002, a tyrosine kinase inhibitor (TKI) that is one of the most potent FLT3 inhibitors discovered to date. Studies using human FLT3/ITD mutant leukemia cell lines revealed the half maximal inhibitory concentration (IC50) for inhibiting FLT3 autophosphorylation is from 100 to 250 pM. The proliferation IC50 for TTT-3002 in these same cells was from 490 to 920 pM. TTT-3002 also showed potent activity when tested against the most frequently occurring FLT3-activating point mutation, FLT3/D835Y, against which many current TKIs are ineffective. These findings were validated in vivo by using mouse models of FLT3-associated AML. Survival and tumor burden of mice in several FLT3/ITD transplantation models is significantly improved by administration of TTT-3002 via oral dosing. Finally, we demonstrated that TTT-3002 is cytotoxic to leukemic blasts isolated from FLT3/ITD-expressing AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from healthy blood and bone marrow donors. Therefore, TTT-3002 has demonstrated preclinical potential as a promising new FLT3 TKI in the treatment of FLT3-mutant AML.
PML-RARA and AML1-ETO are important oncogenic fusion proteins that play a central role in transformation to acute myeloid leukemia (AML). Whether these fusion proteins render the tumor cells with immune evasion properties is unknown. Here we show that both oncogenic proteins specifically downregulate the expression of CD48, a ligand of the natural killer (NK) cell activating receptor 2B4, thereby leading to decreased killing by NK cells. We demonstrate that this process is histone deacetylase (HDAC)-dependent, that it is mediated through the downregulation of CD48 messenger RNA, and that treatment with HDAC inhibitors (HDACi) restores the expression of CD48. Furthermore, by using chromatin immuoprecepitation (ChIP) experiments, we show that AML1-ETO directly interacts with CD48. Finally, we show that AML patients who are carrying these specific translocations have low expression of CD48.
Patients with essential thrombocythemia may carry JAK2 (V617F), an MPL substitution, or a calreticulin gene (CALR) mutation. We studied biologic and clinical features of essential thrombocythemia according to JAK2 or CALR mutation status and in relation to those of polycythemia vera. The mutant allele burden was lower in JAK2-mutated than in CALR-mutated essential thrombocythemia. Patients with JAK2 (V617F) were older, had a higher hemoglobin level and white blood cell count, and lower platelet count and serum erythropoietin than those with CALR mutation. Hematologic parameters of patients with JAK2-mutated essential thrombocythemia or polycythemia vera were related to the mutant allele burden. While no polycythemic transformation was observed in CALR-mutated patients, the cumulative risk was 29% at 15 years in those with JAK2-mutated essential thrombocythemia. There was no significant difference in myelofibrotic transformation between the 2 subtypes of essential thrombocythemia. Patients with JAK2-mutated essential thrombocythemia and those with polycythemia vera had a similar risk of thrombosis, which was twice that of patients with the CALR mutation. These observations are consistent with the notion that JAK2-mutated essential thrombocythemia and polycythemia vera represent different phenotypes of a single myeloproliferative neoplasm, whereas CALR-mutated essential thrombocythemia is a distinct disease entity.
Mutations in the calreticulin (CALR) gene were recently discovered in patients with essential thrombocythemia (ET) lacking the JAK2V617F and MPLW515 mutations, but no information is available on the clinical correlates. In this series, CALR mutations were found in 15.5% of 576 World Health Organization–defined ET patients, accounting for 48.9% of JAK2 and MPL wild-type (wt) patients. CALR-mutated patients were preferentially male and showed higher platelet count and lower hemoglobin and leukocyte count compared with JAK2- and MPL-mutated patients. Patients carrying the CALR mutation had a lower risk of thrombosis than JAK2- and MPL-mutated patients; of interest, their risk was superimposable to patients who were wt for the above mutations. CALR mutation had no impact on survival or transformation to post-ET myelofibrosis. Genotyping for CALR mutations represents a novel useful tool for establishing a clonal myeloproliferative disorder in JAK2 and MPL wt patients with thrombocytosis and may have prognostic and therapeutic relevance.
Immune thrombocytopenia (ITP) is an autoimmune disease with a complex heterogeneous pathogenesis and a bleeding phenotype that is not necessarily correlated to platelet count. In this study, the platelet function was assessed in a well-defined cohort of 33 pediatric chronic ITP patients. Because regular platelet function test cannot be performed in patients with low platelet counts, 2 new assays were developed to determine platelet function: first, the microaggregation test, measuring in platelets isolated from 10 mL of whole blood the platelet potential to form microaggregates in response to an agonist; second, the platelet reactivity assay, measuring platelet reactivity to adenosine diphosphate (ADP), convulxin (CVX), and thrombin receptor activator peptide in only 150 μL of unprocessed whole blood. Patients with a severe bleeding phenotype demonstrated a decreased aggregation potential upon phorbol myristate acetate stimulation, decreased platelet degranulation following ADP stimulation, and a higher concentration of ADP and CVX needed to activate the glycoprotein IIbIIIa complex compared with patients with a mild bleeding phenotype. In conclusion, here we have established 2 functional tests that allow for evaluation of platelet function in patients with extremely low platelet counts (<109). These tests show that platelet function is related to bleeding phenotype in chronic ITP.
Hepcidin controls systemic iron availability, and its excess contributes to the anemia of chronic diseases, the most prevalent anemia in hospitalized patients. We previously reported that heparins are efficient hepcidin inhibitors both in vitro and in vivo, but their anticoagulant activity limits therapeutic use. We studied nonanticoagulant heparins produced by N-acetylation and oxidation/reduction (glycol-split) that lost antithrombin-binding affinity. Four nonanticoagulant heparins inhibited hepcidin expression in hepatic HepG2 cells and primary hepatocytes. The 2 most potent ones used in mice suppressed liver hepcidin expression and serum hepcidin in 6 hours, with a significant decrease of spleen iron. This occurred also in lipopolysaccharide (LPS)-treated animals that mimic inflammation, as well as after chronic 1-week treatments, without evident adverse effects on coagulation. Heparin injections increased iron mobilization and facilitated the recovery from the anemia induced by heat-killed Brucella abortus, a model of inflammatory anemia. The heparins were used also in Bmp6–/– mice. A single dose of heparin reduced the already low level of hepcidin of these mice and prevented its induction by LPS. These nonanticoagulant compounds impair bone morphogenetic protein /sons of mothers against decapentaplegic signaling with no evident adverse effect in vivo, even when administered chronically. They may offer a strategy for the treatment of diseases with high hepcidin levels.
The hepatic hormone hepcidin is a key regulator of systemic iron metabolism. Its expression is largely regulated by 2 signaling pathways: the "iron-regulated" bone morphogenetic protein (BMP) and the inflammatory JAK-STAT pathways. To obtain broader insights into cellular processes that modulate hepcidin transcription and to provide a resource to identify novel genetic modifiers of systemic iron homeostasis, we designed an RNA interference (RNAi) screen that monitors hepcidin promoter activity after the knockdown of 19 599 genes in hepatocarcinoma cells. Interestingly, many of the putative hepcidin activators play roles in signal transduction, inflammation, or transcription, and affect hepcidin transcription through BMP-responsive elements. Furthermore, our work sheds light on new components of the transcriptional machinery that maintain steady-state levels of hepcidin expression and its responses to the BMP- and interleukin-6–triggered signals. Notably, we discover hepcidin suppression mediated via components of Ras/RAF MAPK and mTOR signaling, linking hepcidin transcriptional control to the pathways that respond to mitogen stimulation and nutrient status. Thus using a combination of RNAi screening, reverse phase protein arrays, and small molecules testing, we identify links between the control of systemic iron homeostasis and critical liver processes such as regeneration, response to injury, carcinogenesis, and nutrient metabolism.
In this study, we report on 8 compound heterozygotes for mutations in the key erythroid transcription factor Krüppel-like factor 1 in patients who presented with severe, transfusion-dependent hemolytic anemia. In most cases, the red cells were hypochromic and microcytic, consistent with abnormalities in hemoglobin synthesis. In addition, in many cases, the red cells resembled those seen in patients with membrane defects or enzymopathies, known as chronic nonspherocytic hemolytic anemia (CNSHA). Analysis of RNA and protein in primary erythroid cells from these individuals provided evidence of abnormal globin synthesis, with persistent expression of fetal hemoglobin and, most remarkably, expression of large quantities of embryonic globins in postnatal life. The red cell membranes were abnormal, most notably expressing reduced amounts of CD44 and, consequently, manifesting the rare In(Lu) blood group. Finally, all tested patients showed abnormally low levels of the red cell enzyme pyruvate kinase, a known cause of CNSHA. These patients define a new type of severe, transfusion-dependent CNSHA caused by mutations in a trans-acting factor (Krüppel-like factor 1) and reveal an important pathway regulating embryonic globin gene expression in adult humans.
Contraction of blood clots is necessary for hemostasis and wound healing and to restore flow past obstructive thrombi, but little is known about the structure of contracted clots or the role of erythrocytes in contraction. We found that contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral erythrocytes within. The same results were obtained after initiation of clotting with various activators and also with clots from reconstituted human blood and mouse blood. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, were also observed in human arterial thrombi taken from patients. The mechanical nature of this shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from erythrocytes and to compress erythrocytes into a tightly packed array. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and help explain how fibrinolysis is greatly retarded as clots contract.
Graft-versus-host disease (GVHD) is a systemic inflammatory response due to the recognition of major histocompatibility complex disparity between donor and recipient after hematopoietic stem cell transplantation (HSCT). T-cell activation is critical to the induction of GVHD, and data from our group and others have shown that regulatory T cells (Tregs) prevent GVHD when given at the time of HSCT. Using multiphoton laser scanning microscopy, we examined the single cell dynamics of donor T cells and dendritic cells (DCs) with or without Tregs postallogeneic transplantation. We found that donor conventional T cells (Tcons) spent very little time screening host DCs. Tcons formed stable contacts with DCs very early after transplantation and only increased velocity in the lymph node at 20 hours after transplant. We also observed that Tregs reduced the interaction time between Tcons and DCs, which was dependent on the generation of interleukin 10 by Tregs. Imaging using inducible Tregs showed similar disruption of Tcon–DC contact. Additionally, we found that donor Tregs induce host DC death and down-regulate surface proteins required for donor T-cell activation. These data indicate that Tregs use multiple mechanisms that affect host DC numbers and function to mitigate acute GVHD.
The safety and efficacy of reduced-intensity conditioning (RIC) regimens for the treatment of pediatric acute myeloid leukemia is unknown. We compared the outcome of allogeneic hematopoietic cell transplantation in children with acute myeloid leukemia using RIC regimens with those receiving myeloablative-conditioning (MAC) regimens. A total of 180 patients were evaluated (39 with RIC and 141 with MAC regimens). Results of univariate and multivariate analysis showed no significant differences in the rates of acute and chronic graft-versus-host disease, leukemia-free, and overall survival between treatment groups. The 5-year probabilities of overall survival with RIC and MAC regimens were 45% and 48%, respectively (P = .99). Moreover, relapse rates were not higher with RIC compared with MAC regimens (39% vs 39%; P = .95), and recipients of MAC regimens were not at higher risk for transplant-related mortality compared with recipients of RIC regimens (16% vs 16%; P = .73). After carefully controlled analyses, we found that in this relatively modest study population, the data supported a role for RIC regimens for acute myeloid leukemia in children undergoing allogeneic hematopoietic cell transplantation. The data also provided justification for designing a carefully controlled randomized clinical trial that examines the efficacy of regimen intensity in this population.