Evaluation of platelet function in essential thrombocythemia under different analytical conditions.

Background. Studies of platelet aggregation (PA) in essential thrombocythemia (ET) reported contrasting results, likely due to differences in analytical conditions.Objective. We investigated platelet aggregation using different techniques and analytical conditions.Patients and Methods. PA was studied by light-transmission aggregometry (LTA) in platelet-rich plasma (PRP) and impedance aggregometry in PRP and whole blood (WB). ADP, collagen, thrombin receptor activating peptide (TRAP-14) and adrenaline were used as agonists. Since ET patients (n = 41) were on treatment with aspirin (100 mg/d), healthy controls (n = 29) were given aspirin (100 mg/d) for 5 days before testing: therefore, thromboxane A2-independent PA was tested in all subjects. Blood samples were collected in citrate (C) [low Ca2+] or lepirudin (L) [physiological Ca2+]; platelet count was adjusted to 250 x 109/L in a set of C-PRP (adjusted C-PRP) and left unmodified in the other samples.Results. Results of PA in 17 ET patients who were poor responders to aspirin (high serum thromboxane B2 levels) were not included in the analysis. With LTA, PA in ET was lower than in controls in adjusted C-PRP and normal in native C-PRP and L-PRP. With impedance aggregometry, PA in L-PRP and L-WB tended to be higher in ET than in controls. Platelet serotonin and ADP contents were reduced in ET. The percentages of circulating platelets expressing P-selectin and platelet-leukocyte hetero-aggregates were higher in ET.Conclusions. Analytical conditions dramatically affect in vitro PA of ET patients, which appears defective under the least physiological conditions and normal/supranormal under conditions that are closer to the physiological.

Primary autoimmune neutropenia in adults: case report and review of the literature.

BACKGROUND: Primary autoimmune neutropenia (AIN) is a rare and often unrecognized disorder in adults. STUDY DESIGN AND METHODS: We report the case of a patient referred to our institution for weight loss and severe chronic neutropenia with a negative personal history for severe recurrent infections. RESULTS: The patient was diagnosed with a lung infiltrate, and a bronchoalveolar lavage was positive for Pseudomonas aeruginosa. Antibiotic therapy was performed with resolution of infection, but persistence of neutropenia. Several investigations excluded the most common causes of neutropenia and a marrow trephine showed a maturation arrest of the myeloid lineage. Treatment with granulocyte-colony-stimulating factor (G-CSF) caused a transient increase in neutrophil counts. Based on the mild clinical history and the short-lived increase in neutrophil count after G-CSF, primary AIN was suspected. Intravenous immunoglobulins induced a short-lived increase in neutrophil count; primary AIN was confirmed about 5 months after discharge by direct and indirect granulocyte immunofluorescence tests. The patient was discharged and no further therapy was required for persistent severe neutropenia in the absence of recurrent infections. CONCLUSION: Primary AIN should be considered early in the diagnostic process of isolated neutropenia, to avoid expensive and time-consuming unnecessary diagnostic procedures.

A prognostic model to predict survival after 6 months of ruxolitinib in patients with myelofibrosis.

Ruxolitinib (RUX) is extensively used in myelofibrosis (MF). Despite its early efficacy, most patients lose response over time and, after discontinuation, have a worse overall survival (OS). Currently, response criteria able to predict OS in RUX-treated patients are lacking, leading to uncertainty regarding the switch to second-line treatments. In this study, we investigated predictors of survival collected after 6 months of RUX in 209 MF patients participating in the real-world ambispective observational RUXOREL-MF study (NCT03959371). Multivariable analysis identified the following risk factors: (1) RUX dose <20 mg twice daily at baseline, months 3 and 6 (hazard ratio [HR], 1.79; 95% confidence interval [CI], 1.07-3.00; P = .03), (2) palpable spleen length reduction from baseline ≤30% at months 3 and 6 (HR, 2.26; 95% CI, 1.40-3.65; P = .0009), (3) red blood cell (RBC) transfusion need at months 3 and/or 6 (HR, 1.66; 95% CI, 0.95-2.88; P = .07), and (4) RBC transfusion need at all time points (ie, baseline and months 3 and 6; HR, 2.32; 95% CI, 1.19-4.54; P = .02). Hence, we developed a prognostic model, named Response to Ruxolitinib After 6 Months (RR6), dissecting 3 risk categories: low (median OS, not reached), intermediate (median OS, 61 months; 95% CI, 43-80), and high (median OS, 33 months; 95% CI, 21-50). The RR6 model was validated and confirmed in an external cohort comprised of 40 MF patients. In conclusion, the RR6 prognostic model allows for the early identification of RUX-treated MF patients with impaired survival who might benefit from a prompt treatment shift.

In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method.

Low-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these "non-responders" patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC-MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37-63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8-1222) for EC-ASA, and 823.1(624-1196) ng h/mL (median, 25-75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

Patients with Essential Thrombocythemia may be Poor Responders to Enteric-Coated Aspirin, but not to Plain Aspirin.

Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10-1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

Association of V617F Jak2 mutation with the risk of thrombosis among patients with essential thrombocythaemia or idiopathic myelofibrosis: a systematic review.

INTRODUCTION: Many studies evaluated the association of V617F Jak-2 with the risk of thrombosis in patients with essential thrombocythaemia, but the results of these studies were inconsistent. Few studies evaluated the association of V617F Jak-2 mutation with the risk of thrombosis in patients with idiopathic myelofibrosis. Therefore, we performed a systematic review of the studies that assessed the risk of thrombosis associated with V617F Jak-2 in patients with ET or IM. MATERIALS AND METHODS: We searched MEDLINE and EMBASE databases and reference lists of retrieved articles. Odds ratios (ORs) and 95% confidence intervals (CI) were calculated for each trial and pooled. RESULTS: We included 21 studies involving patients with essential thrombocythaemia and 6 studies patients with idiopathic myelofibrosis. In essential thrombocythaemia patients, V617F Jak-2 was associated with a significant increased risk of thrombosis (OR 1.92, 95% CI 1.45-2.53), both of venous (OR 2.49, 95% CI 1.71-3.61) and arterial (OR 1.77, 95% CI 1.29-2.43) vessels. In idiopathic myelofibrosis patients, the risk of thrombosis associated with V617F Jak-2 tended to be increased (OR 1.76, 95% CI 0.91-3.41). CONCLUSIONS: Our systematic review suggests that V617F Jak-2 increases the risk of thrombosis in essential thrombocythaemia patients by about two fold while its role in idiopathic myelofibrosis patients is uncertain.

Prognostic implications of the European consensus for grading of bone marrow fibrosis in chronic idiopathic myelofibrosis.

Various clinical prognostic scoring systems (PSSs) have been suggested as means of selecting high-risk chronic idiopathic myelofibrosis (CIMF) patients at diagnosis. The WHO has recently proposed strict diagnostic criteria for CIMF, and the European consensus for bone marrow fibrosis (BMF) grading recommends 4 classes. It has been suggested that BMF grading may play a prognostic role in CIMF, but it has never been compared with the other PSSs in the same patients. We tested a prognostic model for overall survival (OS) based on the WHO criteria and BMF grading in 113 consecutive patients with chronic myeloproliferative disorders (98 with CIMF and 15 with postpolycythemic myelofibrosis), and compared the findings with those of PSSs. The results showed that our model is significantly associated with different OSs and, unlike the other PSSs, clearly discriminates the OS of intermediate- and high-risk patients.

Clinical profile of homozygous JAK2 617V>F mutation in patients with polycythemia vera or essential thrombocythemia.

JAK2 617V>F mutation occurs in a homozygous state in 25% to 30% of patients with polycythemia vera (PV) and 2% to 4% with essential thrombocythemia (ET). Whether homozygosity associates with distinct clinical phenotypes is still under debate. This retrospective multicenter study considered 118 JAK2 617V>F homozygous patients (104 PV, 14 ET) whose clinical characteristics were compared with those of 587 heterozygous and 257 wild-type patients. Irrespective of their clinical diagnosis, homozygous patients were older, displayed a higher leukocyte count and hematocrit value at diagnosis, and presented larger spleen volume. Aquagenic pruritus was significantly more common among homozygous PV patients. JAK2 617V>F homozygosity associated with more frequent evolution into secondary myelofibrosis in both PV and ET. After adjustment for sex, age, leukocyte count, and previous thrombosis in a multivariate analysis, homozygous ET patients displayed a significantly higher risk of cardiovascular events (hazard ratio [HR] 3.97, 95% confidence interval [CI] 1.34-11.7; P = .013) than wild-type (HR = 1.0) or heterozygous patients (HR = 1.49). No significant association of JAK2 617V>F homozygosity with thrombosis risk was observed in PV. Finally, JAK2 617V>F homozygous patients were more likely to receive chemotherapy for control of disease. We conclude that JAK2 617V>F homozygosity identifies PV or ET patients with a more symptomatic myeloproliferative disorder and is associated with a higher risk of major cardiovascular events in patients with ET.

Long-term outcome of ph-negative acute lymphoblastic leukaemia in adults: a single centre experience.

BACKGROUND AND OBJECTIVES: In adult acute lymphoblastic leukaemia (ALL), unlike in childhood ALL, the percentage of long-term remitters and survivors has not improved significantly over the last decades. In the present analysis, we describe a series of adult ALL patients consecutively treated with the same regimen in order to analyse prognostic factors and treatment outcome as well as to define new risk-oriented strategies. DESIGN AND METHODS: From 1990 to 1998, 102 newly diagnosed ALL patients were referred to our division, 83 of them were eligible for the present study. Median age was 31 years (range 13-76); 77.1% had B-lineage ALL and 22.9% T-lineage ALL; 36.1% showed associated myeloid markers. All patients received an induction phase treatment, consisting of a 4-week cycle with vincristine, daunorubicin, L-asparaginase and desametasone; the consolidation phase included cyclophosphamide, cytarabine, 6-mercaptopurine and central nervous system (CNS) prophylaxis, followed by three of months maintenance (methotrexate + 6-mercaptopurine), re-induction (4-week cycle with vincristine, adriamicin, desametasone), and 2-year maintenance with methotrexate + 6-mercaptopurine. RESULTS: Complete remission (CR) was achieved in 66 patients (79.5%); 20.5% of patients were resistant. The relapse rate was 60.2%. There were 10 CNS relapses (accounting for 12% of all patients, 15% of all CRs and 20% of all relapses). One patient had an ovarian and 2 had a breast relapse. Eleven patients remained in first continuous CR after chemotherapy. Median overall survival (OS) and disease-free survival (DFS) were 1.8 and 1.0 years, respectively, with a median follow-up of 5.6 years (range 0.3-12.1). Initial white blood cell count

Polycythemia vera in young patients: a study on the long-term risk of thrombosis, myelofibrosis and leukemia.

BACKGROUND AND OBJECTIVES: Polycythemia vera (PV) is unusual in young patients, so that little information is available on long-term clinical evolution in this particular group. The aim of this study was to define the long-term risk of thrombosis, acute leukemia (AL) and myelofibrosis with myeloid metaplasia (MMM) in young PV patients. DESIGN AND METHODS: From 1975 to 2000, 70 PV patients aged less than 50 years were followed for a median time of 14 years (range 2-26). About three quarters were treated with pipobroman. The Kaplan-Meier method and Cox regression were used for survival analysis. The standardized mortality ratio (SMR) was calculated using Italian age/sex specific mortality rates. RESULTS: The risk of thrombosis increased during the observation period, reaching a plateau of 14% at 10 years and was markedly higher in individuals with a previous history of thrombosis (p=0.0023). No patient had progression into AL or MMM before the 9th year of follow-up. Subsequently, five patients (7%) developed AL and five (7%) MMM, with a 20-year cumulative risk of 15% and 10%, respectively. Overall survival at 20 years was 62%, with nine patients dying of progression into AL or MMM, four of vascular events, one of lung cancer, and four of non-PV-related causes. The SMR was 5.3, indicating a mortality significantly higher than that of the general population (p<0.000). INTERPRETATION AND CONCLUSIONS: This long-term retrospective cohort study shows that although the median survival of young patients with PV exceeds 23 years, their life expectancy is markedly lower than that of the general population because of disease evolution into AL or MMM.

A combination of dexamethasone, cyclophosphamide, etoposide, and cisplatin is less toxic and more effective than high-dose cyclophosphamide for peripheral stem cell mobilization in multiple myeloma.

BACKGROUND AND OBJECTIVES: The purpose of this study was to compare the efficacy and toxicity of two regimens for peripheral blood stem cell (PBSC) mobilization in multiple myeloma (MM) patients. DESIGN AND METHODS: From 1995 to 2001, 116 patients were enrolled in two high-dose programs including autologous transplantation, adopting two mobilizing regimens: 61 patients were mobilized with high-dose cyclophosphamide (HD-Cy) at 4 g/m2 (group I), and 55 patients with DCEP (dexamethasone, cyclophosphamide, etoposide, and cisplatin) (group II), both followed by granulocyte colony-stimulating factor (G-CSF 5 mg/Kg/day) started 48 hours after chemotherapy. RESULTS: The median number of CD34+ cells harvested was similar in the two groups (5.9 vs 5.82x106 cells/kg). The target of at least 4x106 cells/kg was reached in a higher percentage of patients in the DCEP group (75 vs 59%) (p=0.05). The proportion of poor mobilizers (<2x106 CD34+ cells/kg) was 21% with HD-Cy and 13% with DCEP (P=NS). In group I, 10 patients (16%) required packed red cell transfusions, 5 patients (8%) platelet support, and the majority of patients (87%) had a neutrophil count below 500/mL, whereas none did so in group II (p=0.0009, p=0.01, p=0.0009, respectively). Neutropenia-related fever occurred in 18% of patients in group I versus 0% in group II (p=0.0005). WHO grade >II extra-hematologic toxicities (microhematuria, cystitis, infections) were seen in 8 patients (13%) of group I vs 0 in group II (p=0.007). INTERPRETATION AND CONCLUSIONS: DCEP is a better tolerated and more effective regimen than HD-Cy for peripheral stem cell mobilization in MM patients assigned to high-dose therapy programs.