National Medical Research Center for Hematology

The therapeutic landscape of chronic myeloid leukemia (CML) has profoundly changed over the past 7 years. Most patients with chronic phase (CP) now have a normal life expectancy. Another goal is achieving a stable deep molecular response (DMR) and discontinuing medication for treatment-free remission (TFR). The European LeukemiaNet convened an expert panel to critically evaluate and update the evidence to achieve these goals since its previous recommendations. First-line treatment is a tyrosine kinase inhibitor (TKI; imatinib brand or generic, dasatinib, nilotinib, and bosutinib are available first-line). Generic imatinib is the cost-effective initial treatment in CP. Various contraindications and side-effects of all TKIs should be considered. Patient risk status at diagnosis should be assessed with the new EUTOS long-term survival (ELTS)-score. Monitoring of response should be done by quantitative polymerase chain reaction whenever possible. A change of treatment is recommended when intolerance cannot be ameliorated or when molecular milestones are not reached. Greater than 10% BCR-ABL1 at 3 months indicates treatment failure when confirmed. Allogeneic transplantation continues to be a therapeutic option particularly for advanced phase CML. TKI treatment should be withheld during pregnancy. Treatment discontinuation may be considered in patients with durable DMR with the goal of achieving TFR.

Owing to increasing resistance and the limited arsenal of new antibiotics, especially against Gram-negative pathogens, carefully designed antibiotic regimens are obligatory for febrile neutropenic patients, along with effective infection control. The Expert Group of the 4(th) European Conference on Infections in Leukemia has developed guidelines for initial empirical therapy in febrile neutropenic patients, based on: i) the local resistance epidemiology; and ii) the patient's risk factors for resistant bacteria and for a complicated clinical course. An 'escalation' approach, avoiding empirical carbapenems and combinations, should be employed in patients without particular risk factors. A 'de-escalation' approach, with initial broad-spectrum antibiotics or combinations, should be used only in those patients with: i) known prior colonization or infection with resistant pathogens; or ii) complicated presentation; or iii) in centers where resistant pathogens are prevalent at the onset of febrile neutropenia. In the latter case, infection control and antibiotic stewardship also need urgent review. Modification of the initial regimen at 72-96 h should be based on the patient's clinical course and the microbiological results. Discontinuation of antibiotics after 72 h or later should be considered in neutropenic patients with fever of unknown origin who are hemodynamically stable since presentation and afebrile for at least 48 h, irrespective of neutrophil count and expected duration of neutropenia. This strategy aims to minimize the collateral damage associated with antibiotic overuse, and the further selection of resistance.

Platelet microparticles (PMPs) are small vesicles released from blood platelets upon activation. The procoagulant activity of PMPs has been previously mainly characterized by their ability to bind coagulation factors VIII and Va in reconstructed systems. It can be supposed that PMPs can contribute to the development of thrombotic complications in the pathologic states associated with the increase of their blood concentration. In this study, we compared procoagulant properties of calcium ionophore A23187-activated platelets and PMPs using several in-vitro models of hemostasis. Surface densities of phosphatidylserine, CD61, CD62P and factor X bound per surface area unit were determined by flow cytometry. They were 2.7-, 8.4-, 4.3-, and 13-fold higher for PMPs than for activated platelets, respectively. Spatial clot growth rate (V(clot)) in the reaction-diffusion experimental model and endogenous thrombin potential (ETP) were determined in plasma, which was depleted of phospholipid cell surfaces by ultra-centrifugation and supplemented with activated platelets or PMPs at different concentrations. Both V(clot) and ETP rapidly increased with the increase of PMP or platelet concentration until saturation was reached. The plateau values of V(clot) and ETP for activated platelets and PMPs were similar. In both assays, the procoagulant activity of one PMP was almost equal to that of one activated platelet despite at least two-orders-of-magnitude difference in their surface areas. This suggests that the PMP surface is approximately 50- to 100-fold more procoagulant than the surface of activated platelets.

BACKGROUND: Familial Mediterranean fever, mevalonate kinase deficiency (also known as the hyperimmunoglobulinemia D syndrome), and the tumor necrosis factor receptor-associated periodic syndrome (TRAPS) are monogenic autoinflammatory diseases characterized by recurrent fever flares. METHODS: We randomly assigned patients with genetically confirmed colchicine-resistant familial Mediterranean fever, mevalonate kinase deficiency, or TRAPS at the time of a flare to receive 150 mg of canakinumab subcutaneously or placebo every 4 weeks. Patients who did not have a resolution of their flare received an add-on injection of 150 mg of canakinumab. The primary outcome was complete response (resolution of flare and no flare until week 16). In the subsequent phase up to week 40, patients who had a complete response underwent a second randomization to receive canakinumab or placebo every 8 weeks. Patients who underwent a second randomization and had a subsequent flare and all other patients received open-label canakinumab. RESULTS: At week 16, significantly more patients receiving canakinumab had a complete response than those receiving placebo: 61% vs. 6% of patients with colchicine-resistant familial Mediterranean fever (P<0.001), 35% versus 6% of those with mevalonate kinase deficiency (P=0.003), and 45% versus 8% of those with TRAPS (P=0.006). The inclusion of patients whose dose was increased to 300 mg every 4 weeks yielded a complete response in 71% of those with colchicine-resistant familial Mediterranean fever, 57% of those with mevalonate kinase deficiency, and 73% of those with TRAPS. After week 16, an extended dosing regimen (every 8 weeks) maintained disease control in 46% of patients with colchicine-resistant familial Mediterranean fever, 23% of those with mevalonate kinase deficiency, and 53% of those with TRAPS. Among patients who received canakinumab, the most frequently reported adverse events were infections (173.3, 313.5, and 148.0 per 100 patient-years among patients with colchicine-resistant familial Mediterranean fever, those with mevalonate kinase deficiency, and those with TRAPS, respectively), with a few being serious infections (6.6, 13.7, and 0.0 per 100 patient-years). CONCLUSIONS: In this trial, canakinumab was effective in controlling and preventing flares in patients with colchicine-resistant familial Mediterranean fever, mevalonate kinase deficiency, and TRAPS. (Funded by Novartis; CLUSTER ClinicalTrials.gov number, NCT02059291 .).

Therapeutic options for chronic myelogenous leukemia (CML) resistant to 400 to 600 mg imatinib are limited. Escalating imatinib doses may overcome resistance. Dasatinib, a significantly more potent inhibitor of BCR-ABL, is safe and effective in this population. Patients with imatinib-resistant chronic-phase (CP) CML were randomized 2:1 to 140 mg dasatinib (n=101) or 800 mg imatinib (n=49). With a median follow up of 15 months, complete hematologic responses were observed in 93% and 82% of patients receiving dasatinib and high-dose imatinib (P=.034), respectively. Dasatinib resulted in higher major cytogenetic response rates (52%) than high-dose imatinib (33%) (P=.023); this included complete cytogenetic response in 40% and 16% (P=.004). Major molecular responses were also more frequent with dasatinib (16% versus 4%; P=0.038). Treatment failure (hazard ratio [HR], 0.16; P<.001) and progression-free survival (HR, 0.14; P<.001) both favored dasatinib. Superficial edema (42% versus 15%) and fluid retention (45% versus 30%) were more prevalent with imatinib; pleural effusion was more common with dasatinib (17% versus 0%). Grade 3 to 4 nonhematologic toxicity was minimal. Cytopenias were more frequent and severe with dasatinib. Dasatinib represents a safe and effective therapy for CP-CML resistant to conventional imatinib doses with improved cytogenetic and molecular response rates and progression-free survival relative to high-dose imatinib.

BACKGROUND: Current hemophilia treatment involves frequent intravenous infusions of clotting factors, which is associated with variable hemostatic protection, a high treatment burden, and a risk of the development of inhibitory alloantibodies. Fitusiran, an investigational RNA interference (RNAi) therapy that targets antithrombin (encoded by SERPINC1), is in development to address these and other limitations. METHODS: In this phase 1 dose-escalation study, we enrolled 4 healthy volunteers and 25 participants with moderate or severe hemophilia A or B who did not have inhibitory alloantibodies. Healthy volunteers received a single subcutaneous injection of fitusiran (at a dose of 0.03 mg per kilogram of body weight) or placebo. The participants with hemophilia received three injections of fitusiran administered either once weekly (at a dose of 0.015, 0.045, or 0.075 mg per kilogram) or once monthly (at a dose of 0.225, 0.45, 0.9, or 1.8 mg per kilogram or a fixed dose of 80 mg). The study objectives were to assess the pharmacokinetic and pharmacodynamic characteristics and safety of fitusiran. RESULTS: No thromboembolic events were observed during the study. The most common adverse events were mild injection-site reactions. Plasma levels of fitusiran increased in a dose-dependent manner and showed no accumulation with repeated administration. The monthly regimen induced a dose-dependent mean maximum antithrombin reduction of 70 to 89% from baseline. A reduction in the antithrombin level of more than 75% from baseline resulted in median peak thrombin values at the lower end of the range observed in healthy participants. CONCLUSIONS: Once-monthly subcutaneous administration of fitusiran resulted in dose-dependent lowering of the antithrombin level and increased thrombin generation in participants with hemophilia A or B who did not have inhibitory alloantibodies. (Funded by Alnylam Pharmaceuticals; ClinicalTrials.gov number, NCT02035605 .).

Patients with chronic myeloid leukemia in chronic phase (CML-CP) resistant/intolerant to ≥2 tyrosine kinase inhibitors (TKIs) are at high risk of experiencing poor outcomes because of disease biology and inadequate efficacy and/or safety of current therapies. Asciminib, a first-in-class BCR-ABL1 inhibitor Specifically Targeting the ABL Myristoyl Pocket (STAMP), has the potential to overcome resistance/intolerance to approved TKIs. In this phase 3, open-label study, patients with CML-CP previously treated with ≥2 TKIs were randomized (2:1) to receive asciminib 40 mg twice daily vs bosutinib 500 mg once daily. Randomization was stratified by major cytogenetic response (MCyR) status at baseline. The primary objective was to compare the major molecular response (MMR) rate at week 24 for asciminib vs bosutinib. A total of 233 patients were randomized to asciminib (n = 157) or bosutinib (n = 76). Median follow-up was 14.9 months. The MMR rate at week 24 was 25.5% with asciminib and 13.2% with bosutinib. The difference in MMR rate between treatment arms, after adjusting for MCyR at baseline, was 12.2% (95% confidence interval, 2.19-22.30; 2-sided P = .029). Fewer grade ≥3 adverse events (50.6% vs 60.5%) and adverse events leading to treatment discontinuation (5.8% vs 21.1%) occurred with asciminib than with bosutinib. The study showed a superior efficacy of asciminib compared with that of bosutinib, together with a favorable safety profile. These results support the use of asciminib as a new therapy in patients with CML-CP who are resistant/intolerant to ≥2 prior TKIs. This trial was registered at www.clinicaltrials.gov as #NCT03106779.

BACKGROUND: This intercontinental study aimed to study gram-negative rod (GNR) resistance in hematopoietic stem cell transplantation (HSCT). METHODS: GNR bacteremias occurring during 6 months post-HSCT (February 2014-May 2015) were prospectively collected, and analyzed for rates and risk factors for resistance to fluoroquinolones, noncarbapenem anti-Pseudomonas β-lactams (noncarbapenems), carbapenems, and multidrug resistance. RESULTS: Sixty-five HSCT centers from 25 countries in Europe, Australia, and Asia reported data on 655 GNR episodes and 704 pathogens in 591 patients (Enterobacteriaceae, 73%; nonfermentative rods, 24%; and 3% others). Half of GNRs were fluoroquinolone and noncarbapenem resistant; 18.5% carbapenem resistant; 35.2% multidrug resistant. The total resistance rates were higher in allogeneic HSCT (allo-HSCT) vs autologous HSCT (auto-HSCT) patients (P < .001) but similar in community-acquired infections. Noncarbapenem resistance and multidrug resistance were higher in auto-HSCT patients in centers providing vs not providing fluoroquinolone prophylaxis (P < .01). Resistance rates were higher in southeast vs northwest Europe and similar in children and adults, excluding higher fluoroquinolone- and β-lactam/β-lactamase inhibitor resistance rates in allo-HSCT adults. Non-Klebsiella Enterobacteriaceae were rarely carbapenem resistant. Multivariable analysis revealed resistance risk factors in allo-HSCT patients: fluoroquinolone resistance: adult, prolonged neutropenia, breakthrough on fluoroquinolones; noncarbapenem resistance: hospital-acquired infection, breakthrough on noncarbapenems or other antibiotics (excluding fluoroquinolones, noncarbapenems, carbapenems), donor type; carbapenem resistance: breakthrough on carbapenem, longer hospitalization, intensive care unit, previous other antibiotic therapy; multidrug resistance: longer hospitalization, breakthrough on β-lactam/β-lactamase inhibitors, and carbapenems. Inappropriate empiric therapy and mortality were significantly more common in infections caused by resistant bacteria. CONCLUSIONS: Our data question the recommendation for fluoroquinolone prophylaxis and call for reassessment of local empiric antibiotic protocols. Knowledge of pathogen-specific resistance enables early appropriate empiric therapy. Monitoring of resistance is crucial. CLINICAL TRIALS REGISTRATION: NCT02257931.

OBJECTIVE: An increased risk of tuberculosis has been documented in humans treated with tumor necrosis factor alpha (TNFalpha)-neutralizing agents. In murine models, impaired signaling by TNF causes exacerbation of both acute and chronic infection associated with aberrant granuloma formation and maintenance. This study was undertaken to investigate immune modulation in the setting of TNF neutralization in primary and latent tuberculosis in a non-human primate model. METHODS: Cynomolgus macaques 4 years of age or older were infected with Mycobacterium tuberculosis and subjected to clinical, microbiologic, immunologic, and radiographic examinations. Monkeys were classified as having active or latent disease 6-8 months after infection, based on clinical criteria. Monkeys used in acute infection studies were randomized to receive either adalimumab (prior to and during infection) or no treatment. Monkeys with latent infection that were randomized to receive TNF-neutralizing agent were given either an inhibitor of soluble TNF, recombinant methionyl human soluble TNF receptor I (p55-TNFRI), or adalimumab. Control monkeys with latent infection were given no treatment or saline. Data from previously studied monkeys with active or latent disease were also used for comparison. RESULTS: Administration of TNF-neutralizing agents prior to M tuberculosis infection resulted in fulminant and disseminated disease by 8 weeks after infection. Neutralization of TNF in latently infected cynomolgus macaques caused reactivation in a majority of animals as determined by gross pathologic examination and bacterial burden. A spectrum of dissemination was noted, including extrapulmonary disease. Surprisingly, monkeys that developed primary and reactivation tuberculosis after TNF neutralization had similar granuloma structure and composition to that of control monkeys with active disease. TNF neutralization was associated with increased levels of interleukin-12, decreased levels of CCL4, increased chemokine receptor expression, and reduced mycobacteria-induced interferon-gamma production in blood but not in the affected mediastinal lymph nodes. Finally, the first signs of reactivation often occurred in thoracic lymph nodes. CONCLUSION: These findings have important clinical implications for determining the mechanism of TNF neutralization-related tuberculosis.

The history of the discovery and study of chemical oscillations and waves is presented from the very first accidental observations up to the systematic design of chemical oscillators. Special emphasis is devoted to the long-term debate over the possibility of pure chemical oscillations, i.e., concentration oscillations in homogeneous closed systems.

Fluorescence light microscopy (LM) has many advantages for the study of cell organization. Specimen preparation is easy and relatively inexpensive, and the use of appropriate tags gives scientists the ability to visualize specific proteins of interest. LM is, however, limited in resolution, so when one is interested in ultrastructure, one must turn to electron microscopy (EM), even though this method presents problems of its own. The biggest difficulty with cellular EM is its limited utility in localizing macromolecules of interest while retaining good structural preservation. We have built a cryo-light microscope stage that allows us to generate LM images of vitreous samples prepared for cryo-EM. Correlative LM and EM allows one to find areas of particular interest by using fluorescent proteins or vital dyes as markers within vitrified samples. Once located, the sample can be placed in the EM for further study at higher resolution. An additional benefit of the cryo-LM stage is that photobleaching is slower at cryogenic temperatures (-140 degrees C) than at room temperature.

BACKGROUND: Preclinical studies demonstrate synergism between cancer immunotherapy and local radiation, enhancing anti-tumor effects and promoting immune responses. BI1361849 (CV9202) is an active cancer immunotherapeutic comprising protamine-formulated, sequence-optimized mRNA encoding six non-small cell lung cancer (NSCLC)-associated antigens (NY-ESO-1, MAGE-C1, MAGE-C2, survivin, 5T4, and MUC-1), intended to induce targeted immune responses. METHODS: We describe a phase Ib clinical trial evaluating treatment with BI1361849 combined with local radiation in 26 stage IV NSCLC patients with partial response (PR)/stable disease (SD) after standard first-line therapy. Patients were stratified into three strata (1: non-squamous NSCLC, no epidermal growth factor receptor (EGFR) mutation, PR/SD after ≥4 cycles of platinum- and pemetrexed-based treatment [n = 16]; 2: squamous NSCLC, PR/SD after ≥4 cycles of platinum-based and non-platinum compound treatment [n = 8]; 3: non-squamous NSCLC, EGFR mutation, PR/SD after ≥3 and ≤ 6 months EGFR-tyrosine kinase inhibitor (TKI) treatment [n = 2]). Patients received intradermal BI1361849, local radiation (4 × 5 Gy), then BI1361849 until disease progression. Strata 1 and 3 also had maintenance pemetrexed or continued EGFR-TKI therapy, respectively. The primary endpoint was evaluation of safety; secondary objectives included assessment of clinical efficacy (every 6 weeks during treatment) and of immune response (on Days 1 [baseline], 19 and 61). RESULTS: Study treatment was well tolerated; injection site reactions and flu-like symptoms were the most common BI1361849-related adverse events. Three patients had grade 3 BI1361849-related adverse events (fatigue, pyrexia); there was one grade 3 radiation-related event (dysphagia). In comparison to baseline, immunomonitoring revealed increased BI1361849 antigen-specific immune responses in the majority of patients (84%), whereby antigen-specific antibody levels were increased in 80% and functional T cells in 40% of patients, and involvement of multiple antigen specificities was evident in 52% of patients. One patient had a partial response in combination with pemetrexed maintenance, and 46.2% achieved stable disease as best overall response. Best overall response was SD in 57.7% for target lesions. CONCLUSION: The results support further investigation of mRNA-based immunotherapy in NSCLC including combinations with immune checkpoint inhibitors. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT01915524 .

To evaluate the relationship between osteoblast differentiation and bioenergetics, cultured primary osteoblasts from fetal rat calvaria were grown in medium supplemented with ascorbate to induce differentiation. Before ascorbate treatment, the rate of glucose consumption was 320 nmol. h(-1). 10(6) cells(-1), respiration was 40 nmol. h(-1). 10(6) cells(-1), and the ratio of lactate production to glucose consumption was approximately 2, indicating that glycolysis was the main energy source for immature osteoblasts. Ascorbate treatment for 14 days led to a fourfold increase in respiration, a threefold increase in ATP production, and a fivefold increase in ATP content compared with that shown in immature cells. Confocal imaging of mitochondria stained with a transmembrane potential-sensitive vital dye showed that mature cells possessed abundant amounts of high-transmembrane-potential mitochondria, which were concentrated near the culture medium-facing surface. Acute treatment of mature osteoblasts with metabolic inhibitors showed that the rate of glycolysis rose to maintain the cellular energy supply constant. Thus progressive differentiation coincided with changes in cellular metabolism and mitochondrial activity, which are likely to play key roles in osteoblast function.

Type 1 (non-neuronopathic) Gaucher disease was the first lysosomal storage disorder for which an effective enzyme replacement therapy was developed and it has become a prototype for treatments for related orphan diseases. There are currently four treatment options available to patients with Gaucher disease, nevertheless, almost 25% of Type 1 Gaucher patients do not gain timely access to therapy because of delays in diagnosis after the onset of symptoms. Diagnosis of Gaucher disease by enzyme testing is unequivocal, but the rarity of the disease and nonspecific and heterogeneous nature of Gaucher disease symptoms may impede consideration of this disease in the differential diagnosis. To help promote timely diagnosis and optimal management of the protean presentations of Gaucher disease, a consensus meeting was convened to develop algorithms for diagnosis and disease management for Gaucher disease. The defect in Gaucher disease is an inherited deficiency of the lysosomal enzyme acid β-glucosidase (glucocerebrosidase, GBA1), which results in the accumulation of glucocerebroside within lysosomes of macrophages. Systemic accumulation of these glycolipid-lipid engorged cells (eponymously known as Gaucher cells, Fig. 1) results in variable combinations of splenomegaly with associated abdominal discomfort; anemia associated with chronic fatigue; bleeding due to thrombocytopenia and/or Gaucher disease-related coagulopathy; hepatomegaly, abnormal tests of liver function; and a diverse pattern of bone disease [1]. Increased susceptibility to infections may result from impaired neutrophil function and neutropenia [2]. Rarely, the lungs, lymphatic system, skin, eyes, kidneys, and the heart are involved and, in the rare neuronopathic forms, neurodegenerative disease results [1]. Gaucher disease is traditionally classified into three broad phenotypic categories: Type 1 (non-neuronopathic disease); Type 2, fulminant neuronopathic disease that is fatal during infancy; and Type 3, chronic neuronopathic disease, that usually results in death in childhood or early adult life [1]. Further distinct phenotypic categories may be recognized within these broad groups. Bone marrow smear and biopsy from a patient with N370S homozygous Gaucher disease showing classical striated macrophages and marrow replacement. Smear: Gaucher cells (×100). Core: bone marrow replacement by Gaucher cells (×20). Type 1 (non-neuronopathic) Gaucher disease accounts for more than 90% all Gaucher disease patients. Its prevalence world wide is 1 in 50,000 to 100,000 but it is as high as ∼1 in 850 in individuals of Ashkenazi heritage [3-6]. The broadest phenotypic spectrum in Gaucher disease with respect to age of onset, rate of progression, and organs affected occurs in Type 1 Gaucher disease [7]. Homozygosity for the N370S mutation is the most common genotype in the Ashkenazim in whom it accounts for ∼70% of all disease alleles. It is associated with atypical presentation in adults or the older patient and is notable for significant skeletal disease despite inconspicuous classical manifestations (splenomegaly, hepatomegaly, anemia, and thrombocytopenia). However, severe disease with classic manifestations presenting in childhood may occur in a minority of N370S homozygous patients [8, 9]. The most common disease allele of GBA1 world wide is L444P mutation, which occurs in the sequence of the closely linked pseudogene; it is believed that gene conversion events lead to L444P mutation in the active gene. It seems likely that the most frequent genotype of Type 1 Gaucher disease in populations of European descent in the world is N370S/L444P. Generally this genotype leads to more severe disease compared with N370S homozygosity [1, 10, 11] (Figs. 2 and 3). Relationship between age of presentation at extent of splenomegaly (determined by volumetric MRI with normal spleen volume at 0.2% body weight; extent of splenomegaly is indicated by multiple of normal, i.e. ×N). Three most common genotypes in North American Gaucher disease patient populations are depicted: N370S/N370S, N370S/L444P, and N370S/84G ins G). The results show that the majority of older N370S homozygous patients harbor no, or minimal, splenomegaly). (a and b). Comparison of severity of hepatomegaly and bone disease (Herman score [9]) in patients with intact spleen and those with prior splenectomy stratified for GBA1 gene genotype. The numbers of patients are depicted at the bottom of the bars. Treatment with macrophage-targeted mannose-terminated glucocerebrosidase enzyme replacement therapy (imiglucerase, Cerezyme®, Genzyme Corporation, Cambridge, MA) is the standard of care for Type 1 Gaucher disease and of non-neuronopathic manifestations of Type 3 Gaucher disease. Evidence from the ICGG Gaucher Registry, indicates that enzyme replacement with imiglucerase and its predecessor alglucerase reverses hematological and visceral manifestations of the disease [12] and reduces the bone marrow burden of Gaucher cells resulting in amelioration of osteopenia, bone pain, risk of bone crises and overall improvement in quality of life [13-16]. Several aspects of bone disease, such as osteonecrosis, osteofibrosis, and lytic lesions cannot be reversed but timely initiation of enzyme therapy reduces the risk of these irreversible complications [8, 17]. Other variants of macrophage-targeted enzyme replacement therapies are undergoing clinical trials: velaglucerase, a human fibroblast-derived enzyme, was recently approved for treatment of Type 1 Gaucher disease [18] and taliglucerase, a plant derived enzyme is in clinical trials [19]. Substrate reduction therapy (Zavesca® miglustat, Actelion Pharmaceuticals, Allschwil, Switzerland) is approved for patients with mild Gaucher disease who are unable to receive enzyme replacement therapy [20, 21]. A more specific and potent inhibitor of glucosylceramide synthesis, eliglustat tartrate is currently in Phase 3 trials having shown impressive efficacy and safety in Phase 2 trials [22, 23]. Prompt diagnosis before the occurrence of irreversible complications underpins the Gaucher disease management model [24]. The early symptoms of Type 1 Gaucher disease tend to reflect the hematological aspects of the disease (splenomegaly, anemia, thrombocytopenia, and bleeding tendency) [25]. Patients with Gaucher disease, therefore, are most likely to be referred to hematologists for diagnosis and management. However, only ∼20% of hematologists/oncologists in one study considered Gaucher disease in differential diagnoses even in the presence of all classical symptoms [8]. Diagnosis may be achieved in high-risk groups, such as individuals of Ashkenazi Jewish ancestry, by opportunistic screening (i.e., those with any of the manifestations of Gaucher disease or those with surrogate indicators of disease, i.e, severe osteoporosis, hyperferritinemia, gallstones, and low HDL cholesterol) and family screening after diagnosis in a family member [26]. The aim of the consensus meeting was to develop algorithms for diagnosis and management based on current understanding of the full clinical spectrum of presentations of Gaucher disease. The authors' combined experience of 362 patients with Gaucher disease revealed a consistent pattern of previous misdiagnoses that included leukemia, immune thrombocytopenia purpura, autoimmune disease, hepatic cirrhosis, idiopathic avascular necrosis, viral disease, idiopathic splenomegaly, and anemia of chronic disease. Misdiagnosis led to complications such as avascular necrosis, osteopenia, liver disease, and bleeding complications and inappropriate procedures such as splenectomy, liver biopsy, and empirical corticosteroid therapy (see Appendix). Malignancy is commonly the first diagnosis entertained in patients who are eventually diagnosed with Gaucher disease [8, 27]. Interestingly, the first patient ever described with Gaucher disease by Dr Philippe Gaucher in 1882 was believed to harbor malignancy affecting the spleen. In one study, the diagnosis most frequently considered was hematologic malignancy (leukemia 65%, lymphoma 36%, multiple myeloma 22%, chronic granulocytic leukemia 14%) [8]. It should be noted that the risk of hematological malignancies in Gaucher disease is increased, especially of multiple myeloma [9, 28-31]. The life-time risk of multiple myeloma in Gaucher disease is higher than 25-fold compared with the general population [9, 29-32]. In patients of Ashkenazi ancestry, the frequency of Gaucher disease is ∼1 in 800 while hematologic malignancies are much less frequent at ∼1 in 2,500 [33]. Therefore, in this ethnic group, it is prudent to test for Gaucher disease as a first-line investigation, in any patient presenting with splenomegaly and cytopenia. It is important to keep in mind that the most common genotype in this ethnic group, homozygosity for N370S mutation is often characterized by mild cytopenia and splenomegaly that escape initial detection. Therefore, presence of conditions associated with Gaucher disease should alert the clinician, i.e., hyperferritinemia, low HDL cholesterol, premature gallstones or osteoporosis, and gammopathy. In the non-Ashkenazi populations Gaucher disease is markedly less frequent (∼1 in 40,000) compared with hematologic malignancies. In this setting, it would be appropriate to consider Gaucher disease in the differential diagnosis after malignancies have been ruled out. In this setting, bone marrow biopsy is usually performed; it should become routine to search for Gaucher cells as well as for evidence of hematological malignancies. The above considerations form the rationale for the diagnostic algorithm stratified by ethnicity (Figs. 4 and 5). Portal hypertension due to advanced liver disease from well-known etiologies must be ruled out before application of the algorithm. Diagnosis algorithm for individuals of Ashkenazi origin. Diagnosis of Gaucher disease should be considered in any individual of Ashkenazi Jewish ancestry presenting with mild, moderate or severe splenomegaly. When splenomegaly is absent, Gaucher disease should be considered in the presence of thrombocytopenia (even if mild), bleeding tendency, unexplained stable hyperferritinemia with normal transferrin saturation, or increased inflammatory markers. *In patients with bleeding diatheses, coagulopathies such as factor XI deficiency common in Askenazim [34] should be excluded. Diagnostic algorithm for individuals of non-Ashkenazi Jewish origin. Compared with Gaucher disease, malignancy is likely to be the more frequent cause of splenomegaly in this population. In this setting it is reasonable to perform bone marrow biopsy in initial investigations. The finding of Gaucher cells in bone marrow aspirate will suggest Gaucher disease although Gaucher disease and malignancy are not mutually exclusive. Pseudo-Gaucher cells have also been observed in malignant conditions in the absence of Gaucher disease [35]. In developing these diagnostic algorithms, the authors focused on splenomegaly, as a key presenting sign since it is present in the vast majority of Gaucher patients [10]. In the ICGG Registry 87% of patients have splenomegaly in excess of five times normal (median spleen volume = 15.2 × normal) [6]. However, Gaucher disease may occur without splenomegaly and the absence of splenomegaly does not exclude Gaucher disease (Fig. 2). This is often the case in older adults homozygous for the N370S mutation in whom splenomegaly is mild and may not be present [9]. Splenomegaly is defined precisely by volumetric measurement of the spleen by MRI, CT or ultrasound scanning followed by conversion to weight (1 mL equals 1 gm) as multiple of the normal spleen size, i.e., 0.2% body weight [36]. Splenectomy in patients with Gaucher disease is associated with aggressive disease in the liver, skeleton, and the lungs [37] (Fig. 3). Therefore, it should become mandatory to test for Gaucher disease in any patient being considered for splenectomy when the cause of splenomegaly has not been established. Assessment of splenomegaly in Gaucher disease should be combined with an examination of spleen parenchyma. There is almost 20% incidence of focal splenic defects in Gaucher disease and its incidence increases with increasing spleen size [38]. These lesions may represent focal collections of Gaucher cells or areas of infarction and have been a source of misdiagnosis as splenic neoplasms. The diagnostic test for Gaucher disease is the demonstration of low acid β-glucosidase activity in peripheral blood leukocytes (normal range 2.1–5.3 μmol/l/hr) [39]. The assay is performed in blood leukocytes using a fluorescent substrate, 4-methyumbelliferone β-glucoside. The test requires 10 mL EDTA blood sample shipped at ambient temperature by overnight delivery to the lab. Molecular analysis of GBA1 gene is complicated by presence of highly homologous pseudogene that harbors several mutations, which if present in the active gene leads to Gaucher disease. A number of techniques have been developed to circumvent potential problems arising from this situation in order to analyze only the active gene sequences uncontaminated by pseudogene sequences [40]. A negative screen for common GBA1 mutations does not exclude Gaucher disease (see Discussion). Therefore, undertaking sequencing of the entire coding region of GBA1 gene is recommended in patients strongly suspected of harboring Gaucher disease when a screen for common mutations is negative [40]. Mutation analysis of the GBA1 gene may provide some prognostic information although there is considerable variation of disease severity among patients harboring an identical GBA1 genotype [7] (Fig. 2). Knowledge of the GBA1 mutation in a proband also facilitates family screening for genetic counseling purposes since heterozygote carriers cannot be reliably identified by enzyme assays. Routine bone marrow examination is not necessary for diagnosis. It has been discouraged in the diagnosis of Gaucher disease, due to availability of less invasive and robust enzymatic test and the fact that it may cause bleeding complications due to thrombocytopenia and coagulopathy. Many patients report having had this procedure usually before Gaucher disease had been considered. Care should be exercised in the interpretation of bone marrow samples as false-negative results are common especially upon examination of bone marrow aspirate as opposed to bone marrow biopsy. Conversely, the presence of pseudo-Gaucher cells may lead to erroneous diagnosis [35]. Pseudo-Gaucher cells have been found in diverse conditions such as multiple myeloma [41], myelodysplasia and myelodysplastic syndromes [42], chronic myeloid leukemia [43], pulmonary tuberculosis [44], mycobacterioses [45], and sickle cell disease [46]. Occasionally bone marrow biopsy may be indicated in a patient with Gaucher disease suspected of a superimposed hematologic disorder, i.e., myelofibrosis, multiple myeloma, or other hematologic malignancy. Several serum proteins are consistently elevated in Gaucher disease, with some being used as biomarkers for routine monitoring, i.e., angiotensin-converting enzyme (ACE), tartrate-resistant acid phosphatase (TRAP), chitotriosidase, and a chemokine, CCL18 [47, 48]. The finding of elevated levels of one or more of these markers is never sufficient to diagnose Gaucher disease since these markers may be elevated in other Other are common in Gaucher disease, such as low low serum and and elevated [9, a diagnosis of Gaucher disease is the should be on of all disease (Fig. to disease to for enzyme replacement and to develop and of life and a severity score using one of the that are in the are of in treatment skeletal is especially important since it is the of most irreversible which occur without of neuronopathic Gaucher disease has been described In non-neuronopathic disease examination should also a search for symptoms and of and peripheral (see Discussion). A in the management and of Gaucher disease is that it is a chronic disease that is at variable and leads to [9, treatment and to the manifestations of Gaucher disease are based on the experience with alglucerase and imiglucerase enzyme replacement therapy which is a for all other treatment Assessment of to treatment is derived from a body of evidence The heterogeneous and chronic nature of Type 1 Gaucher disease requires an disease management model with respect to enzyme and in disease be achieved by therapy before irreversible complications occur compared with a It is that there is a for imiglucerase therapy Therefore, for patients in whom disease is a i.e., moderate to severe disease those with complications such as and pulmonary the initial of imiglucerase is usually body weight in a Patients may to after initial disease (Fig. In patients with such as pulmonary enzyme therapy followed by other appropriate therapy should be adult patients with less severe disease, initial of body is a prudent patients may not treatment because have mild or even disease such patients are usually of N370S homozygous genotype. These patients should be to of disease that treatment be before symptoms or irreversible occurs (Fig. It is important to that patients may be harbor significant disease manifestations such as splenomegaly, and These patients should receive treatment to disease and algorithm. of patients therapy imiglucerase as have been a may be with to that disease does not Treatment must be one treatment will all patients. The diagnosis and management algorithms are likely to be to the majority of patients with Gaucher disease despite there being more than mutations in the GBA1 gene that to vast phenotypic mutations ins 2 and for of disease in patients of Ashkenazi Jewish ancestry and in patients of non-Ashkenazi descent [1]. In the most disease genotype is the homozygous being associated with severe childhood onset and a high risk of [1, 10, There is phenotypic among patients with GBA1 genotype as well as among affected (Fig. 2). is only in by GBA1 gene mutations and the important of has been but Gaucher disease may be associated with manifestations that escape an with the enzymatic defect and lysosomal storage of The understanding of the of GBA1 mutations and of glucosylceramide accumulation in macrophages and its on other cell severe has been described in some patients with Type 1 Gaucher disease, and this has its non-neuronopathic in Type 1 is believed to from within by gain of function mutations in GBA1 that lead to N370S is such a or accumulation of rare but fatal of Type 1 is pulmonary hypertension that occurs in and may of cells in the disease Other of complications peripheral and hematologic malignancies [9, The increased risk of multiple myeloma and in Type 1 Gaucher disease has been to chronic immune macrophages Type 3 Gaucher disease a distinct is associated with homozygosity for mutation that disease, and and [40]. is the most severe of Type 2 disease, a that is in GBA1 these have the of Gaucher disease. into the of Gaucher disease have revealed important of cell other than in of Gaucher disease the the management of Gaucher disease and the for affected patients has a and it an of to the in the of phenotypic of Gaucher disease, the and understanding of the of enzyme replacement therapy with imiglucerase into algorithms for management. Further is with in gene therapy and therapy for mutations that result in defects A is therapy that to to therapy for with [22, this is likely to represent a significant to current therapy and to some of the in of diagnosis using blood will access to testing and timely diagnosis. The to develop diagnostic and disease management algorithms was in of the for and early diagnosis of Gaucher disease among hematologists [8]. Genzyme an to the consensus of in and the management of Gaucher disease convened in to and clinical with the of developing diagnosis and management algorithms for Gaucher disease. out of by or of by on such as to individual to also by to from the Gaucher Registry of patients world The 10 experience of Gaucher disease presentations and in of the world in an to of the a of the of misdiagnosis from case are included in Appendix). the first meeting of of misdiagnoses in Gaucher disease, presenting sign and symptoms in Gaucher patients from clinical and those described in the on these key diagnostic These the for the of diagnostic The algorithms for diagnosis and management of Gaucher disease developed and and between The authors are to for with of the or in this are those of the from and had to a body for several Further not performed at this the age of was diagnosed with Gaucher disease for N370S family screening a diagnosis of Gaucher disease in The patient had mild splenomegaly, and was not the patient was that had mild Type 1 Gaucher disease and not the patient had an onset of severe bone in the (Fig. of liver volume × and spleen volume × avascular and of the of childhood MRI revealed avascular the and The patient had score of (normal range to the age of enzyme the age of replacement. (a and b). MRI showing avascular in the in a homozygous for N370S mutation with and cytopenia There is infarction in the that eventually replacement. There is of marrow in the due to marrow normal marrow MRI is depicted in that in childhood due to avascular was not When the patient was diagnosed with Gaucher disease as an adult family severe bone disease was not identified due to on and hematologic which indicated only mild disease. A was for chronic liver disease due to the finding of and elevated liver negative and The patient had chronic bone in diagnosed as the age of was diagnosed with of severe anemia and due to thrombocytopenia Bone marrow examination revealed almost replacement by Gaucher A diagnosis of Type 1 Gaucher disease was glucocerebrosidase revealed chronic liver disease and bone marrow The patient for with bone disease, liver disease, and eventually bone marrow a routine a Ashkenazi was found to have and hyperferritinemia was blood and was for an with early onset disease. A liver biopsy revealed Gaucher cells (Fig. The diagnosis of Type 1 Gaucher disease was by low acid β-glucosidase activity in blood leukocytes and homozygosity for the N370S Further revealed splenomegaly and biopsy showing accumulation of of cells Gaucher in the to normal A was diagnosed with Gaucher disease on liver biopsy performed for of and are common in Gaucher disease. A was followed for for severe thrombocytopenia, diagnosed as immune thrombocytopenia was with without The patient had a previous and developed pain, which was to be to the was to because of Further revealed splenomegaly, advanced avascular of the and Bone marrow biopsy revealed Gaucher Misdiagnosis as led to chronic corticosteroid therapy and eventually to avascular and A patient a with symptoms due to However, there was and bone marrow revealed Gaucher The diagnosis was by low acid β-glucosidase activity in blood Further splenomegaly, elevated chitotriosidase, normal and Gaucher disease may be associated with symptoms. In this with A and abdominal examination and appropriate have the of Gaucher disease liver and bone marrow biopsy. procedures a high risk of bleeding in this

We have analyzed 1448 patients with acquired aplastic anemia grafted between 2005 and 2009, and compared outcome of identical sibling (n=940) versus unrelated donor (n=508) transplants. When compared to the latter, sibling transplants were less likely to be performed beyond 180 days from diagnosis (39% vs. 85%), to have a cytomegalovirus negative donor/recipient status (15% vs. 23%), to receive antithymocyte globulin in the conditioning (52% vs. 61%), and more frequently received marrow as a stem cell source (60% vs. 52%). Unrelated donor grafts had significantly more acute grade II-IV (25% vs. 13%) and significantly more chronic graft-versus-host disease (26% vs. 14%). In multivariate analysis, the risk of death of unrelated donor grafts was higher, but not significantly higher, compared to a sibling donor (P=0.16). The strongest negative predictor of survival was the use of peripheral blood as a stem cell source (P<0.00001), followed by an interval of diagnosis to transplant of 180 days or more (P=0.0005), patient age 20 years or over (P=0.0005), no antithymocyte globulin in the conditioning (P=0.003), and donor/recipient cytomegalovirus sero-status, other than negative/negative (P=0.04). In conclusion, in multivariate analysis, the outcome of unrelated donor transplants for acquired aplastic anemia, is currently not statistically inferior when compared to sibling transplants, although patients are at greater risk of acute and chronic graft-versus-host disease. The use of peripheral blood grafts remains the strongest negative predictor of survival.

Dasatinib is the most potent BCR-ABL inhibitor, with 325-fold higher potency than imatinib against unmutated BCR-ABL in vitro. Studies have demonstrated the benefits of dasatinib 70 mg twice daily in patients with accelerated-phase chronic myeloid leukemia intolerant or resistant to imatinib. A phase 3 study compared the efficacy and safety of dasatinib 140 mg once daily with the current twice-daily regimen. Here, results from the subgroup with accelerated-phase chronic myeloid leukemia (n = 317) with a median follow-up of 15 months (treatment duration, 0.03-31.15 months) are reported. Among patients randomized to once-daily (n = 158) or twice-daily (n = 159) treatment, rates of major hematologic and cytogenetic responses were comparable (major hematologic response, 66% vs 68%; major cytogenetic response, 39% vs 43%, respectively). Estimated progression-free survival rates at 24 months were 51% and 55%, whereas overall survival rates were 63% versus 72%. Once-daily treatment was associated with an improved safety profile. In particular, significantly fewer patients in the once-daily group experienced a pleural effusion (all grades, 20% vs 39% P < .001). These results demonstrate that dasatinib 140 mg once daily has similar efficacy to dasatinib 70 mg twice daily but with an improved safety profile. This trial is registered at www.clinicaltrials.gov as #CA180-035.

Eliglustat tartrate is an investigational oral substrate reduction therapy for Gaucher disease type 1 that is pharmacologically distinct from intravenous enzyme replacement therapy. Eliglustat tartrate improved clinical manifestations in patients who received 50 or 100 mg twice daily for 1 year during an open-label phase 2 study (Blood. 2010;116(6):893-899). We report further improvements after 2 years of treatment in 20 patients (11 females, 9 males; mean age, 33 years) with baseline splenomegaly and thrombocytopenia and/or anemia. Statistically significant (P < .001) percentage improvements from baseline occurred in platelet count (mean ± SD, 81% ± 56%), hemoglobin level (20% ± 15%), spleen volume (-52% ± 11%), and liver volume (-24% ± 13%). Mean platelet count increased ∼ 50 000/mm(3). Mean hemoglobin level increased 2.1 g/dL overall and 3.1 g/dL in 10 patients with baseline anemia. Organ volume reductions were greatest in patients with severe baseline organomegaly. Seventeen (85%) patients met established therapeutic goals for ≥ 3 of the 4 parameters. Lumbar spine bone mineral density increased 7.8% ± 10.6% (P = .01) and T-score 0.6 ± 0.8 (P = .012), with major gains in osteoporotic and osteopenic patients. Magnetic resonance imaging assessment showed that bone marrow infiltration by Gaucher cells was decreased (8/18 patients) or stable (10/18 patients). No safety-related trends emerged during 2 years of treatment. This multisite, open-label, single-arm phase 2 study is registered at www.clinicaltrials.gov as NCT00358150.

The detection of multi-resistant bacterial pathogens, particularly those to carbapenemases, in leukemic and stem cell transplant patients forces the use of old or non-conventional agents as the only remaining treatment options. These include colistin/polymyxin B, tigecycline, fosfomycin and various anti-gram-positive agents. Data on the use of these agents in leukemic patients are scanty, with only linezolid subjected to formal trials. The Expert Group of the 4(th) European Conference on Infections in Leukemia has developed guidelines for their use in these patient populations. Targeted therapy should be based on (i) in vitro susceptibility data, (ii) knowledge of the best treatment option against the particular species or phenotype of bacteria, (iii) pharmacokinetic/pharmacodynamic data, and (iv) careful assessment of the risk-benefit balance. For infections due to resistant Gram-negative bacteria, these agents should be preferably used in combination with other agents that remain active in vitro, because of suboptimal efficacy (e.g., tigecycline) and the risk of emergent resistance (e.g., fosfomycin). The paucity of new antibacterial drugs in the near future should lead us to limit the use of these drugs to situations where no alternative exists.

Eliglustat tartrate (Genz-112638), a specific inhibitor of glucosylceramide synthase, is under development as an oral substrate reduction therapy for Gaucher disease type 1 (GD1). A multinational, open-label, single-arm phase 2 study of 26 GD1 patients (16 female, 10 male; mean age, 34 years) evaluated the efficacy, safety, and pharmacokinetics of eliglustat tartrate administered twice daily by mouth at 50- or 100-mg doses based on plasma drug concentrations. Entry criteria required splenomegaly with thrombocytopenia and/or anemia. The composite primary efficacy end point required improvement after 52 weeks in at least 2 of these 3 disease manifestations and was met by 77% (95% confidence interval [CI] = 58%-89%) of all patients and 91% (95% CI = 72%-98%) of the 22 patients completing 52 weeks. Statistically significant improvements occurred in mean hemoglobin level (1.62 g/dL; 95% CI =1.05-2.18 g/dL), platelet count (40.3%; 95% CI = 23.7-57.0 g/dL), spleen volume (-38.5%; 95% CI = -43.5%--33.5%), liver volume (-17.0%; 95% CI = -21.6%-12.3%), and lumbar spine bone mineral density (0.31 Z-score; 95% CI = 0.09-0.53). Elevated biomarkers (chitotriosidase; chemokine CCL18; angiotensin-converting enzyme; tartrate-resistant acid phosphatase) decreased by 35% to 50%. Plasma glucosylceramide and ganglioside GM3 normalized. Eliglustat tartrate was well tolerated: 7 mild, transient adverse events in 6 patients were considered treatment-related. Individual pharmacokinetics varied; mean time to maximal observed concentration was 2.3 hours and mean half-life was 6.8 hours. Eliglustat tartrate appears to be a promising oral treatment for GD1.

Although high-dose methotrexate (HD-MTX) is the most effective drug against primary CNS lymphomas (PCNSL), outcome-determining variables related to its administration schedule have not been defined. The impact on toxicity and outcome of the area under the curve (AUC(MTX)), dose intensity (DI(MTX)) and infusion rate (IR(MTX)) of MTX and plasmatic creatinine clearance (CL(crea)) was investigated in a retrospective series of 45 PCNSL patients treated with three different HD-MTX-based combinations. Anticonvulsants were administered in 31 pts (69%). Age >60 years, anticonvulsant therapy, slow IR(MTX) (</=800 mgm(-2)h(-1)), and reduced DI(MTX) (</=1000 mgm(-2)wk(-1)) were significantly correlated with low AUC(MTX) values. Seven patients (16%) experienced severe toxicity, which was independently associated with slow CL(crea). A total of 18 (40%) patients achieved complete remission after chemotherapy, which was independently associated with slow CL(crea). In all, 22 patients were alive at a median follow-up of 31 months, with a 3-year OS of 40+/-9%; slow CL(crea) and AUC(MTX) >1100 micromol hl(-1) were independently associated with a better survival. Slow CL(crea) and high AUC(MTX) are favourable outcome-determining factors in PCNSL, while slow CL(crea) is significantly related to higher toxicity. AUC(MTX) significantly correlates with age, anticonvulsant therapy, IR(MTX), and DI(MTX). These findings, which seem to support the choice of an MTX dose >/=3 gm(-2) in a 4-6-h infusion, every 3-4 weeks, deserve to be assessed prospectively in future trials. MTX dose adjustments in patients with fast CL(crea) should be investigated.