Determinant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid-derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.
This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had three treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared rFVIII and rFVIIIFc pharmacokinetics. Endpoints included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 h) was extended 1.5-fold versus rFVIII (12.4 h; P<.001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with one injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1-2 times per week. This study is registered at www.clinicaltrials.gov, ID: NCT01181128.
ACE910 is a recombinant humanized bispecific antibody that binds to activated factor IX and factor X and mimics the cofactor function of factor VIII (FVIII). This first-in-human study examined the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of ACE910 in healthy male adults. A total of 40 Japanese and 24 Caucasian subjects were randomized to receive a single subcutaneous injection of ACE910 (Japanese: 0.001, 0.01, 0.1, 0.3, or 1 mg/kg; Caucasian: 0.1, 0.3, or 1 mg/kg; n = 6 per dose group) or placebo (n = 2 per dose group). ACE910 exhibited a linear PK profile and had a half-life of approximately 4 to 5 weeks. In FVIII-neutralized plasma, ACE910 shortened activated partial thromboplastin time and increased peak height of thrombin generation in a dose-dependent manner. All adverse events were non-serious and did not lead to any subject’s withdrawal. Neither clinical findings nor laboratory abnormalities indicating hypercoagulability were observed. Two of 48 subjects receiving ACE910 (1 Japanese and 1 Caucasian) were positive for anti-ACE910 antibodies (anti-drug antibodies; ADA). One subject tested positive for ADA both before and after ACE910 administration, whereas the other became ADA-positive after receiving ACE910. The PK and PD profiles of ACE910 were similar in healthy Japanese and Caucasian subjects, and suggest that ACE910 will be an effective and convenient prophylactic treatment for hemophilia A. This trial was registered at http://www.clinicaltrials.jp (JapicCTI-121934).
A global Phase 3 study evaluated the pharmacokinetics, efficacy and safety of recombinant fusion protein linking coagulation factor IX with albumin (rIX-FP) in 63 previously treated male patients (12-61 years) with severe hemophilia B (FIX activity ≤ 2%). The study included 2 groups: Group 1 patients received routine prophylaxis once every 7 days for 26 weeks, followed by either 7-, 10- or 14-day prophylaxis regimen for a mean of 50, 38, or 51 weeks, respectively; Group 2 patients received on-demand treatment for bleeding episodes for 26 weeks and then switched to a 7 day prophylaxis regimen for a mean of 45 weeks. The mean terminal half-life of rIX-FP was 102 hours, 4.3-fold longer than previous factor IX (FIX) treatment. Patients maintained a mean trough of 20 and 12 IU/dL FIX activity on prophylaxis with rIX-FP 40 IU/kg weekly and 75 IU/kg every 2 weeks, respectively. There was a 100% reduction in median annualized spontaneous bleeding rate (AsBR) and 100% resolution of target joints when subjects switched from on-demand to prophylaxis treatment with rIX-FP (P <0.0001). The median AsBR was 0.00 for all prophylaxis regimens. Overall, 98.6% of bleeding episodes were treated successfully, including 93.6% that were treated with a single injection. No patient developed an inhibitor and no safety concerns were identified. These results indicate that rIX-FP is safe and effective for preventing and treating bleeding episodes in patients with hemophilia B at dosing regimens of 40 IU/kg weekly and 75 IU/kg every 2 weeks. Clinicaltrials.gov (NCT0101496274).
The dramatic success of tyrosine kinase inhibitors (TKIs) has led to the widespread perception that chronic myeloid leukemia (CML) has become another chronic disease, where lifelong commitment to pharmacological control is the paradigm. Recent trials demonstrate that some CML patients who have achieved stable deep molecular response can safely cease their therapy without relapsing (treatment free remission; TFR). Furthermore, those who are unsuccessful in their cessation attempt can safely re-establish remission after restarting their TKI therapy. Based on the accumulated data on TFR we propose that it is now time to change our approach for the many CML patients who have achieved a stable deep molecular response on long-term TKI therapy. Perhaps half of these patients could successfully achieve TFR if offered the opportunity. For many of these patients ongoing therapy is impairing quality of life and imposing a heavy financial burden while arguably achieving nothing. This recommendation is based on the evident safety of cessation attempts and TFR in the clinical trial setting. We acknowledge that there are potential risks associated with cessation attempts in wider clinical practice, but this should not deter us. Instead we need to establish criteria for safe and appropriate TKI cessation. Clinical trials will enable us to define the best strategies to achieve TFR, but clinicians need guidance today about how to approach this issue with their patients. We outline circumstances in which it would be in the patient’s best interest to continue TKI, as well as criteria for a safe TFR attempt.
A revision of the nearly 8 year old WHO classification of the lymphoid neoplasms and the accompanying monograph is being published. It reflects a consensus among hematopathologists, geneticists and clinicians regarding both updates to current entities as well as the addition of a limited number of new provisional entities. The revision clarifies the diagnosis and management of lesions at the very early stages of lymphomagenesis, refines the diagnostic criteria for some entities, details the expanding genetic/molecular landscape of numerous lymphoid neoplasms and their clinical correlates, and refers to investigations leading to more targeted therapeutic strategies. The major changes are reviewed with an emphasis on the most important advances in our understanding that impact our diagnostic approach, clinical expectations and therapeutic strategies for the lymphoid neoplasms.
The inborn errors of heme biosynthesis, the porphyrias, are 8 genetically distinct metabolic disorders that can be classified as “acute hepatic,” “hepatic cutaneous,” and “erythropoietic cutaneous” diseases. Recent advances in understanding their pathogenesis and molecular genetic heterogeneity have led to improved diagnosis and treatment. These advances include DNA-based diagnoses for all the porphyrias, new understanding of the pathogenesis of the acute hepatic porphyrias, identification of the iron overload-induced inhibitor of hepatic uroporphyrin decarboxylase activity that causes the most common porphyria, porphyria cutanea tarda, the identification of an X-linked form of erythropoietic protoporphyria due to gain-of-function mutations in erythroid-specific 5-aminolevulinate synthase (ALAS2), and new and experimental treatments for the erythropoietic prophyrias. Knowledge of these advances is relevant for hematologists because they administer the hematin infusions to treat the acute attacks in patients with the acute hepatic porphyrias, perform the chronic phlebotomies to reduce the iron overload and clear the dermatologic lesions in porphyria cutanea tarda, and diagnose and treat the erythropoietic porphyrias, including chronic erythrocyte transfusions, bone marrow or hematopoietic stem cell transplants, and experimental pharmacologic chaperone and stem cell gene therapies for congenital erythropoietic protoporphyria. These developments are reviewed to update hematologists on the latest advances in these diverse disorders.
In the present study, we evaluated the efficacy and safety of rituximab in combination with standard doxorubicin, bleomycin, vinblastine, and dacarbazine (RABVD) in patients with classical Hodgkin lymphoma (cHL). In this phase 2 study, patients with chemotherapy-naive, advanced-stage cHL were treated with rituximab 375 mg/m(2) weekly for 6 weeks and standard ABVD for 6 cycles. The primary outcome was event-free survival (EFS) at 5 years. Eighty-five patients were enrolled, of whom 78 were eligible. With a median follow-up duration of 68 months (range, 26-110), and based on an intent-to-treat analysis, the 5-year EFS and overall survival rates were 83% and 96%, respectively. The 5-year EFS for patients with stage III/IV cHL was 82%. Furthermore, the 5-year EFS for patients with an International Prognostic Score of 0-2 was 88% and for those with a score of > 2, it was 73%. The most frequent treatment-related grade 3 or 4 adverse events were neutropenia (23%), fatigue (9%), and nausea (8%). Our results demonstrate that the addition of rituximab to ABVD is safe and has a promising clinical activity in patients with advanced-stage cHL. These data are currently being confirmed in a multicenter randomized trial.
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.
Risk factors for deep-vein thrombosis have been shown not to be always the same as for pulmonary embolism. A well-known example is the factor V Leiden (FVL) paradox: the FVL mutation poses a clearly higher risk for deep-vein thrombosis (DVT) than for pulmonary embolism. We aimed to expand this paradox and therefore present risk estimates for several established risk factors for DVT and pulmonary embolism separately. When such separate risk estimates could not be retrieved from the literature, we calculated these risks in our own data, a large population-based case-control study on venous thrombosis (the MEGA study). Our results showed that the FVL paradox can be broadened (ie, the risk factors oral contraceptive use, pregnancy, puerperium, minor leg injuries, and obesity have an effect comparable with FVL). Furthermore, we found that pulmonary conditions, such as chronic obstructive pulmonary disease, pneumonia, and sickle cell disease, were risk factors with an opposite effect: a higher risk of pulmonary embolism, but little or no effect on DVT. These findings suggest that pulmonary embolism and DVT may not always have the same etiology, and encourage unraveling this phenomenon in further studies.