HEMATOTHESIS

Cytogenetic and genomic assessment of selected lymphoproliferative disorders

Summary

Chronic mature B-cell lymphoproliferative disorders, i.e. B-cell chronic lymphocytic leukaemia and plasma cell dyscrasias such as multiple myeloma, have a variable disease course. Clinical staging systems and several biological parameters have been used to estimate tumour burden and predict prognosis. In addition, cytogenetic aberrations have prognostic significance and are therefore investigated in the routine evaluation of these diseases. In this doctoral study, we evaluated available techniques, which can be applied to detect cytogenetic abnormalities in the routine investigation of chronic lymphocytic leukaemia and multiple myeloma. Next, we characterised cytogenetic entities, in particular translocations involving immunoglobulin genes and the proto-oncogenes BCL2 and MYC and investigated clonal evolution in chronic lymphocytic leukaemia.

(BELG J HEMATOL 2014;5(1):25–30)

A phase I/II single centre study of haploidentical transplantation combined with G-GSF, or GM-CSF, and escalating DLI in high-risk patients with no matched donors

Summary

In 1999, we decided to start a phase I/II study of haploidentical transplantation for high-risk patients. The aim of the work was to implement a strategy to accelerate and strengthen the immune reconstitution by using nonspecific manipulation post-transplant and by developing specific strategies directed against viral antigens. The goal was to increase the graft-versus-leukemia effect without inducing or aggravating the deleterious graft-versus-host disease. The conditioning regimen, adapted to our group of patients, remained the same throughout. Importantly, the first recruited patients were in refractory disease, over time we were referred less advanced patients (complete remission 2 or more). There were 45 patients, all at high-risk, among which 27 were in refractory relapse. We questioned the importance of post-transplant growth factors policy and the influence of donor lymphocyte infusion. Because of the conditioning, transplant-related mortality was low at 3 months, but thereafter changed unfavourably when using granulocyte macrophage-colony stimulating factors in an increased incidence of acute graft-versus-host disease. As a whole the long-term survival of the patients was poor (18%) but improved a lot when transplanted patients were in complete remission (leukaemia-free survival of 39% at five years). Regarding the use of growth factors and donor lymphocyte infusion, granulocyte macrophage-colony stimulating factors with donor lymphocyte infusion induced a very high transplant-related mortality due to a high rate of severe graft-versus-host disease, while the combination of granulocyte colony-stimulating factors and a moderate dose of donor lymphocyte infusion was much safer but didn’t overcome the high relapse rate in refractory patients. The combination of granulocyte colony-stimulating factors and donor lymphocyte infusion might nonetheless be sufficient to decrease the infection rate in patients transplanted in complete remission. The use of granulocyte macrophage-colony stimulating factors leads to an unacceptable lethal graft-versus-host disease rate. The 39% at five years leukaemia-free survival in patients in complete remission compares favourably with what can be achieved with matched unrelated donors in complete remission 2 or more.

(BELG J HEMATOL 2013;4(4): 151–160)

The effects of VEGFA/VEGFR interference on cell survival and angiogenesis in acute myeloid leukaemia: preclinical and clinical studies

Summary

The research described in this thesis aimed to explore the various mechanisms by which vascular endothelial growth factor A promotes acute myeloid leukaemia progression via autocrine and/or paracrine mechanisms, e.g. angiogenesis. Special attention was focused on new potential small-molecule-inhibitors and antibodies interfering with vascular endothelial growth factor/vascular endothelial growth factor receptor signalling. We showed that interference with vascular endothelial growth factor A/vascular endothelial growth factor receptor signalling induces cell death in (paediatric) acute myeloid leukaemia blasts and primitive cells. Furthermore, we studied angiogenesis in bone marrow of acute myeloid leukaemia patients and identified different morphology patterns, related to treatment outcome.

(BELG J HEMATOL 2013;4(3): 112–114)

Functional assessment of haemophilic arthropathy with three-dimensional gait analysis

Summary

In patients with haemophilia, the long-term consequences of repeated haemarthrosis include joint cartilage damage and irreversible chronic arthropathy, resulting in severe impairments in locomotion. Quantifying the extent of joint damage is of paramount importance in order to prevent disease progression and compare the efficacy of treatment strategies, such as prophylaxis. Here we summarise the results of several studies establishing three-dimensional gait analysis as an innovative approach to evaluate functionally haemophilic arthropathy. This work also provides new insights into the understanding of the biome-chanical consequences of haemophilic arthropathy.

(BELG J HEMATOL 2013;4(2):72–76)

Quantification and characterisation of microvesicles: Applications in hereditary spherocytosis, type-II heparin-induced thrombocytopenia and cancer

Summary

Microvesicles (MVs) are sub-micron-size cellular fragments released by eukaryotic cells following activation or apoptosis. Their diameter ranges between 30 and 1000 nm. Micro-vesicles are thought to play a major role in cellular cross-talk, inflammation, thrombosis and angiogenesis. As potential disease biomarkers, MV measurement and characterisation in biological fluids could also reveal new diagnostic and/or prognostic information in human disease. In this work:

• We developed and validated an easy-to-use and useful quality control parameter for MV analysis by flow cytometry (FCM), the most frequently used technique to study MVs.
• We developed and validated a reproducible MV quantification method by FCM in whole blood in order to avoid preanalytical concerns of plasma assays (i.e. loss of MVs by centrifugation and lack of standardisation in centrifugation methods).
• We showed that this method could contribute to the diagnosis of hereditary spherocytosis (HS), a haemolytic anemia characterised by a release of MVs and unexplained occurrence of venous and arterial thrombosis after splenectomy.
• We developed and validated a high sensitive sizing atomic force microscopy (AFM) method.
• We characterised tumour cell-derived MVs released by cultured breast cancer cells MDA-MB 231 (Cells) by FCM, Transmission Electron Microscopy, AFM and Thrombin Generation Assay.
• Finally, we developed a platelet microparticle generation assay (PMPGA), a test which reproduces the in vivo type II heparin-induced thrombocytopenia (HIT) reaction. We showed that this assay, presented at least similar performances in comparison to the current biological reference, i.e. ¹⁴C-Serotonin Release Assay. As flow cytometry is widespread available, PMPGA

(BELG J HEMATOL 2012;3:157–160)

Adoptive immunotherapy for viral infections after allogeneic stem cell transplantation

Summary

On the 22^nd of March 2011, M.L. Zandvliet defended his thesis entitled ‘Adoptive immunotherapy for viral infections after allogeneic stem cell transplantation’ at the University of Leiden, The Netherlands. The research described in this thesis was supervised by professor H.-J. Guchelaar, PharmD, professor J.H.F. Falkenburg, MD, and Dr. P. Meij, PhD. The most important findings of his thesis research are summarised in this report.

(BELG J HEMATOL 2012;3:68–70)

Development of a clinical leukaemia vaccine using dendritic cells loaded with the Wilms’ tumour (WT1) gene product

Summary

Dendritic cell (DC)-based vaccination holds promise as an adjuvant immunotherapy for many cancers. The Wilms’ tumour (WT1) protein is overexpressed in most types of leukaemia and in many solid tumours. Therefore, WT1 could be regarded as a broadly applicable tumour-associated antigen in DC-based immunotherapy. In this thesis, we pursued the aim to enhance antileukemic immune response by the activation of WT1-specific T cells. We developed – from bench to bedside – a therapeutic vaccine of DC loaded with WT1 for patients with acute myeloid leukaemia (AML).

(BELG J HEMATOL 2012;3:24–6)