Development of fludarabine formulations in the treatment of chronic lymphocytic leukemia

Fludarabine is an antineoplastic agent used in the treatment of hematological malignancies, particularly chronic lymphocytic leukemia (CLL) and indolent B-cell lymphoma. Because of its immunosuppressive effects, fludarabine has been added to reduced intensity conditioning regimens. The oral formulation of fludarabine has become widely available. Pharmacokinetic studies have shown that an oral dose of 40 mg/m2/d would provide systemic drug exposure similar to the standard intravenous (IV) dose of 25 mg/m2/d. The oral dose can be taken once daily without any dietary restrictions. Dose adjustments are mandatory in patients with renal impairment to avoid increased toxicity. Several noncomparative trials in previously untreated and treated patients with CLL have shown that treatment with the oral formulation demonstrates similar efficacy compared to historical control groups treated with the IV formulation. The tolerability profile of oral fludarabine seems similar to that of the IV formulation. Myelosuppression and infectious complications are the most frequently reported adverse events. Gastrointestinal toxicity is more frequent with the oral formulation, but is usually of mild or moderate severity. Although oral fludarabine makes treatment more convenient, health care workers must be aware of the compliance behavior of each patient

High CD33-antigen loads in peripheral blood limit the efficacy of gemtuzumab ozogamicin |(Mylotarg®) treatment in acute myeloid leukemia patients

Gemtuzumab ozogamicin (Mylotarg®) induces remission in approximately 30% of relapsed AML patients. We previously demonstrated that gemtuzumab infusion results in near-complete CD33 saturation in peripheral blood, and that saturating gemtuzumab levels result in continuous binding and internalization of gemtuzumab due to renewed CD33 expression. We now demonstrate that a high CD33-antigen load in peripheral blood is an independent adverse prognostic factor, likely due to peripheral consumption of gemtuzumab. Indeed, CD33 saturation in bone marrow is significantly reduced (40-90% saturation) as compared with CD33 saturation in corresponding peripheral blood samples (>90%). In vitro, such reduced CD33 saturation levels were strongly related with reduced cell kill. Apparently, high CD33-antigen loads in blood consume gemtuzumab and thereby limit its penetration into bone marrow. Consequently, CD33 saturation in bone marrow is reduced, which hampers efficient cell kill. Therefore, gemtuzumab should be administered at higher or repeated doses, or, preferably, after reduction of the leukemic cell burden by classical chemotherapy

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

BCR/ABL- CD34(+)HLA-DR- progenitor cells in early chronic phase, but not in more advanced phases, of chronic myelogenous leukemia are polyclonal

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) translocation and BCR/ABL gene rearrangement which occur in a pluripotent hematopoietic progenitor cell. Ph-negative (Ph-) hematopoiesis can be restored in vivo after treatment with -interferon or intensive chemotherapy, suggesting that normal stem and progenitor cells coexist with the Ph+ clone. We have previously shown that Ph- progenitors are highly enriched in the CD34(+)HLA-DR- fraction from early chronic phase (ECP) CML patients. Previous studies have suggested that the Ph-translocation represents a secondary clonal hit occurring in an already clonally mutated Ph- progenitor or stem cells, leaving the unanswered question whether Ph- CD34(+)HLA-DR- progenitors are normal. To show the clonal nature of Ph- CD34(+)HLA-DR- CML progenitors, we have compared the expression of BCR/ABL mRNA with X-chromosome inactivation patterns (HUMARA) in mononuclear cells and in CD34(+)HLA-DR+ and CD34(+)HLA-DR- progenitors in marrow and blood obtained from 11 female CML patients (8 in chronic phase and 3 in accelerated phase [AP] disease). Steady-state marrow-derived BCR/ABL mRNA-, CD34(+)HLA-DR- progenitors had polyclonal X-chromosome inactivation patterns in 2 of 2 patients. The same polyclonal pattern was found in the progeny of CD34(+)HLA-DR- derived long-term culture-initiating cells. Mobilization with intensive chemotherapy induced a Ph-, BCR/ABL mRNA- and polyclonal state in the CD34(+)HLA-DR- and CD34(+)HLA-DR+ progenitors from 2 ECP patients. In a third ECP patient, polyclonal CD34(+) cells could only be found in the first peripheral blood collection. In contrast to ECP CML, steady-state marrow progenitors in late chronic phase and AP disease were mostly Ph+, BCR/ABL mRNA+, and clonal. Further, in the majority of these patients, a Ph-, polyclonal state could not be restored despite mobilization with intensive chemotherapy. We conclude from these studies that CD34(+)HLA-DR- cells that are Ph- and BCR/ABL mRNA- are polyclonal and therefore benign. This population is suitable for autografting in CML.status: publishe

Enhancement of granulocyte-endothelial cell adherence and granulocyte-induced cytotoxicity by platelet release products

Complement-stimulated granulocytes adhere to and induce significant 51Cr release from endothelial cells in vitro. Platelets were stimulated to undergo release, and these release products significantly enhanced granulocyte-endothelial cell adherence and granulocyte-induced 51Cr release from endothelial cells. Platelet serotonin appeared to mediate these phenomena because serotonin antagonists blocked both the enhanced endothelial adherence and 51Cr release. In addition, added serotonin mimicked the effect seen with the stimulated platelets upon granulocyte--endothelial cell adherence and cytotoxicity completely. This enhancement appeared to be due to serotonin effects upon both the granulocyte and endothelial cells. These data suggest that a released platelet constituent might modulate in vivo granulocyte-endothelial cell interactions in clinical disorders.status: publishe

The clinical significance of activated lymphocytes in patients with myelodysplastic syndromes: A single centre study of 131 patients

We studied the immune compartment in patients with myelodysplastic syndromes. We show increased surface expression of activation markers (HLA-DR(+), CD57(+), CD28(-), CD62L(-)) on T lymphocytes in blood and bone marrow (n=131). T cell activation was not restricted to any relevant clinical subgroup (FAB, IPSS, cytogenetics) and did not correlate with blood counts or need for treatment. In vitro clonogenic growth of marrow mononuclear cells (n=18) was not influenced by T cells expressing these markers. In addition, using X-chromosome inactivation analysis (n=12) we demonstrate clonal involvement of NK and B cells in half of these patients. We conclude that although activated T lymphocytes can be found in MDS, their role in disease pathogenesis remains unclear in the majority of patients.status: publishe

CD34+ marrow progenitors from MDS patients with high levels of intramedullary apoptosis have reduced expression of alpha4beta1 and alpha5beta1 integrins

Excessive intramedullary apoptosis is central in the pathogenesis of myelodysplastic syndromes (MDS). Growth-inhibiting cytokines, the Fas/FasLigand pathway, and autoreactive cytotoxic T-lymphocytes have been identified to be important proapoptotic factors in MDS. In normal hematopoiesis, alpha4beta1 and alpha5beta1 integrin-mediated interactions between progenitors and fibronectin are critical for progenitor cell survival. In this study, we have used flow cytometry to quantify the expression levels of members of the beta1 integrin family on CD34(+) marrow progenitors in 27 untreated patients with MDS, three with s-AML, and 25 control subjects. In MDS, we observed that nonapoptotic progenitors significantly downregulate cell surface expression levels of alpha4 and beta1 integrin chains compared with healthy controls. Downregulation of alpha4, beta1, and also alpha5 was present in MDS patients with > or =25% apoptotic progenitors, irrespective of their French, American, British subcategory. Reduced cell surface expression levels of alpha4, alpha5, and beta1 did also correlate with decreased in vitro adhesiveness to fibronectin fragments. Therefore, our observations suggest that downregulation of alpha4beta1 and alpha5beta1 integrins on CD34(+) progenitors could be a newly identified proapoptotic mechanism in MDS.status: publishe

The AMUSE-environment: a didactical environment where active multimedia use stimulates expertise

The rapidly evolving information technology domain continuously offers new opportunities for computer aided education (C.A.E.). In a pilot project at the Department of Medical Informatics, we studied the possibilities and the limitations of multimedia systems for computer aided education in medicine. Different specialists paid attention to the medical, didactic, conceptual and technical points of view. From this teamwork originated the AMUSE-environment: an environment where Active Multimedia Use Stimulates Expertise. The AMUSE-environment describes a didactic framework that focuses on knowledge acquisition and application in complex content domains (the transfer and stimulation of expertise). In the context of advanced learning and random access instruction, the active involvement of the user is mandatory. Multimedia systems enhance communication by integrating different media (e.g., text, pictures, audio, video, ...) in one natural, user-friendly environment. The first application that uses the AMUSE-environment is developed in cooperation with the Haematology Department of the University Hospital Gasthuisberg. The haematologists filled the didactical framework with more than 500 full color illustrations and corresponding text and audio. The application gives a complete overview of the morphology of the blood and bone marrow cells.status: publishe