The miR-200c/141-ZEB2-TGFß axis is aberrant in human T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is mostly characterized by aberrant expansion of small- to medium-sized prolymphocytes with a mature post-thymic phenotype, high aggressiveness of the disease and poor prognosis. However, T-PLL is more heterogeneous with a wide range of clinical, morphological, and molecular features, which occasionally impedes the diagnosis. We hypothesized that T-PLL consists of phenotypic and/or genotypic subgroups that may explain the heterogeneity of the disease. Multi-dimensional immuno-phenotyping and gene expression profiling did not reveal clear T-PLL subgroups, and no clear T-cell receptor a or ß CDR3 skewing was observed between different T-PLL cases. We revealed that the expression of microRNA (miRNA) is aberrant and often heterogeneous in T-PLL. We identified 35 miRNA that were aberrantly expressed in T-PLL with miR-200c/141 as the most differentially expressed cluster. High miR- 200c/141 and miR-181a/181b expression was significantly correlated with increased white blood cell counts and poor survival. Furthermore, we found that overexpression of miR-200c/141 correlated with downregulation of their targets ZEB2 and TGFßR3 and aberrant TGFß1- induced phosphorylated SMAD2 (p-SMAD2) and p-SMAD3, indicating that the TGFß pathway is affected in T-PLL. Our results thus highlight the potential role for aberrantly expressed oncogenic miRNA in T-PLL and pave the way for new therapeutic targets in this disease.

PFA-100 monitoring of von Willebrand factor (VWF) responses to desmopressin (DDAVP) and factor VIII/VWF concentrate substitution in von Willebrand disease type 1 and 2.

Dose-response relationship was studied between PFA-100 closure times (PFA CTs) and factor (F)VIII-von Willebrand factor (VWF) parameters in patients with von Willebrand disease (VWD) type 1 and type 2 before and after treatment with DDAVP (n=84) or FVIII/VWF concentrate (n=38). DDAVP treatment of patients with VWD type 1 normalised the PFA CTs by increasing VWF levels to normal. Of the 14 patients with VWD type 2, PFA CTs did not normalize in eight. Haemate-P substitution in patients with VWD type 1 induced a less favourable response as compared to DDAVP, because PFA CTs did not correct in all patients. Of 12 patients with VWD type 2 treated with Haemate-P, six showed a correction of PFA CTs (<250 sec), which correlated with the normalisation of the VWF CB/Ag ratio. In-vitro studies were performed by using whole blood of patients with VWD and adding various amounts of FVIII/VWF concentrate. Addition of Haemate-P induced an increase of the VWF CB/Ag ratio from 0.30 to 0.70 in blood of patients with VWD type 2 with correction of the PFA CTs. Immunate did not result in an increase of VWF CB/Ag ratio in blood of VWD type 2 patients, and the PFA CTs remained prolonged. We conclude that PFA-100 might be an adequate instrument not only for diagnosis but also for monitoring of DDAVP responses and FVIII/VWF substitution of patients with VWD type 1 and 2, but this is dependent upon the type of VWD and the concentrate used.

Large and medium-sized pulmonary artery obstruction does not play a role of primary importance in the etiology of sickle-cell disease-associated pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PHT) occurs in approximately 30% of adult patients with sickle-cell disease (SCD) and is a risk factor for early death. The potential role of pulmonary artery obstruction, whether due to emboli or in situ thrombosis, in the etiology of SCD-related PHT is unknown. METHODS: Consecutive SCD patients were screened for PHT (defined as a tricuspid regurgitant jet flow velocity > or = 2.5 m/s) employing echocardiography and were evaluated for pulmonary artery obstruction with ventilation-perfusion (VQ) scintigraphy. RESULTS: Fifty-three HbSS, 6 HbSbeta(0)-thalassemia, 20 HbSC, and 6 HbSbeta(+)-thalassemia patients were included. The overall prevalence of PHT was 41% in HbSS/HbSbeta(0)-thalassemia patients and 13% in HbSC/HbSbeta(+)-thalassemia patients. High-probability VQ defects (Prospective Investigation of Pulmonary Embolism Diagnosis criteria) were detected in two patients, one of whom had PHT. In HbSS/HbSbeta(0)-thalassemia patients with PHT, 19 patients (86%), 2 patients (9%), and 1 patient (5%) had low-, intermediate-, or high-probability scan results as compared to 30 patients (97%), 1 patient (3%), and 0 patients (0%) in HbSS/HbSbeta(0)-thalassemia patients without PHT (p = 0.31). In HbSC/HbSbeta(+)-thalassemia patients with PHT, 3 patients (100%), 0 patients (0%), and 0 patients (0%) had low-, intermediate-, and a high-probability scan as compared to 19 patients (90%), 1 patient (5%), and 1 patient (5%) in HbSC/HbSbeta(+)-thalassemia patients without PHT (p = 0.86). There were no statistical differences in irregular distribution of the radiopharmaceutical or nonspecific signs associated with PHT between patients with and without PHT. CONCLUSIONS: Although small pulmonary artery obstruction cannot be excluded, large to medium-sized pulmonary artery obstruction is an unlikely primary causative factor in SCD-related PHT.

A new polyadenylation site mutation associated with a mild beta-thalassemia phenotype.

At least 180,000 autochthonous and allochthonous people are carriers of a large spectrum of hemoglobinopathies in The Netherlands. We describe two cases, the first, a 33-year old Surinamese Creole woman, studied because of an intermediate hemolytic anemia; the second, a couple requesting analysis because of a previously diagnosed carrier state in the male partner, while the carrier state in the pregnant female was uncertain.

In vitro studies, pharmacokinetic studies and clinical use of a high purity double virus inactivated FVIII/VWF concentrate (Immunate) in the treatment of von Willebrand disease.

Patients with type 2 and 3 von Willebrand disease (VWD) are treated with factor VIII/VWF concentrate in case of bleeding or surgery. Immunate (Baxter, Vienna, Austria) is a double virus inactivated FVIII/VWF concentrate and is registered in several countries for patients with VWD with reduced FVIII levels. We performed an in vitro, a pharmacokinetic and a clinical study to evaluate Immunate in VWD. In vitro studies showed a significant variation in VWF levels in 9 different batches. The median (range) values (in IU/mL) were 1.10 (0.98-1.30) for FVIII:C, 1.34 (0.95-1.61) for VWF:Ag, 0.60 (0.27-1.08) for VWF:CBA and 0.73 (0.59-0.94) for VWF:RCo. The relatively low VWF activity is mainly due to the lack of high molecular weight multimers (HMWM), as determined by electrophoresis. A pharmacokinetic study showed, based on a content of FVIII:C of 1 U/mL, in vivo recoveries (%) of 106 (56-150) (median and range) for FVIII:C, 105 (62-187) for VWF:Ag, 25 (7-41) for VWF:CBA and 43 (11-76) for VWF:RCo. Half-lives were 14.1 h (7.4-36.9) for FVIII:C, 10.8 h (7.7-26.2) for VWF:Ag, 15.3 h (7.8-44.6) for VWF:CBA and 16.4 h (4.2-26.5) for VWF:RCo. In a clinical study efficacy was determined after infusion given before surgery or dental extractions in ten patients. In two patients the hemostatic response was classified as inadequate. In conclusion, there is a wide variability in VWF concentration and activity in various batches of Immunate. In the clinical study in which the dosage was based on FVIII:C contents of the concentrate, two out of ten patients had an insufficient haemostatic response. Therefore dosing of Immunate dosing should not be based on FVIII:C levels, but should be based on VWF activity of the individual batches. Future studies using a VWF activity-guided dosage regimen have to be performed to establish the efficacy of Immunate in the treatment of von Willebrand disease.

A novel 7.9 kb deletion causing alpha+-thalassaemia in two independent families of Indian origin.

We describe the characterization of a novel 7.9 kb deletion that eliminated one of the duplicated alpha-globin genes, causing an alpha+-thalassaemia phenotype in two independent carriers of Suriname-Indian origin. The molecular characterization of the deletion breakpoint fragment revealed neither involvement of Alu repeat sequences nor the presence of homologous regions prone to recombination, suggesting a non-homologous recombination event. This alpha+-thalassaemia deletion was found to give rise to an atypical haemoglobin H (HbH) disease characterized by a non-transfusion-dependent moderate microcytic hypochromic anaemia in combination with a poly adenylation signal mutation of the alpha-globin gene (alpha2 AATAAA --> AATA-- --).