Research within this main theme deals with the search into the key molecular processes regulating the proliferation and differentiation of myeloid and lymphoid cells (particularly stem cell biology, erythropoiesis, granulopoiesis, lymphocyte development), and aberrations determining malignant transformation (e.g. in murine models and pathogenetic clinical studies). The basic aspects of the program are complemented by research components related to the function and dysfunction and deficiency of the differentiated “end” cells both in physiological conditions and in disease. Specific programs have an extension towards clinical application and involve investigations related to developmental diagnostics and therapeutics (e.g. molecular diagnostics, pharmacogenomics, therapeutic targeting in leukemia as well as stem cell transplantation, gene therapy). Thus the program covers a spectrum from basic towards clinically applied investigations. Traditionally these programs have had their main basis in the Dept. of Hematology of the Erasmus University Medical Center. More recently the pediatric division of hematology-oncology has joined this main theme. The research program is solidly embedded in and interacting with investigators, scientific groups and networks in a broad international context (e.g. cooperative clinical trial groups, European consortia, scientific groups). This holds both for the laboratory parts and the clinical activities.
Theme 1: Regulation of proliferation and differentiation of hematopoietic stem cells
Prof. dr. Delwel, dr. M. Raaijmakers, Prof. dr. I.P. Touw, dr. J.M.J.M. Zijlmans
Research within this subtheme deals with the control of hematopoietic stem cell and progenitor cell fate. One section deals with the role of extrinsic regulators such as hematopoietic growth factors (G-CSF, EPO, SCF) and chemokines (SDF-1, Cb2 ligands, somatostatin) and their spatial organization within specific cellular components of the hematopoietic stem cell niche Other sections deal with stem cell plasticity, homing and aging. The overall aim of these studies is to elucidate the molecular and cellular mechanisms underlying the principle features of hematopoietic stem cells and precursors: self-renewal, lineage commitment and differentiation, proliferation and survival, migration and aging. They provide insights in the control of normal blood cell development that are crucial to our understanding of how these mechanisms may play a role in the pathogenesis of hematopoietic disorders, such as leukopenia, myelodysplasia and leukemia. Furthermore , investigations are anticipated to generate insights into cues, both environmentally and cell-intrinsic, that maintain and expand hematopoietic stem cell numbers and can be exploited for regenerative purposes. Experimental approaches within this theme include e.g., the generation of animal models employing transgenesis and knockout/knocking strategies, advanced cell culture assays for primary hematopoietic stem cell and progenitor cell subsets, retroviral gene transfer technology, isolation and functional/biochemical analysis of protein complexes analysis and advanced live cell (quantitative) imaging technology (e.g., fluorescence resonance energy transfer and fluorescence recovery after photo bleaching).
Theme 2: Transplantation and genetic modification of hematopoietic stem cells
Dr. E. Braakman, dr. J.J. Cornelissen, dr. T. Cupedo, dr. J. W. Gratama
Within this theme there is a longstanding research effort in murine models for human diseases and nonhuman primate models for stem cell biology and transplantation, which is concerned with the manipulation of immune modulation and the development of gene transfer for therapeutic purposes. Hematopoietic stem cell transplantation (SCT) is currently an important therapeutic modality for many malignant hematological disorders, its use for the treatment of metastatic solid tumors is under investigation as well as its development for gene transfer as a therapeutic modality. Alternative stem cell sources (cord blood) and alternative donors (matched unrelated donors) are increasingly used for hematopoietic stem cell transplantation.
Transplant-related morbidity and mortality of allogeneic SCT is still significant due to acute and chronic graft-versus-host disease (GVHD) and opportunistic infections (mainly reactivations of endogenous herpes viruses). A major cause of opportunistic infections is an impaired immune recovery due to deficient thymopoiesis.
Our research focuses on:
- The identification and treatment of patients with an impaired immune recovery after transplantation at high risk for specific progressive viral infections.
- The development of interventions, including cytokine intervention therapy and thymic regenerative cellular therapy to improve thymopoiesis and immune recovery after transplantation.
- The development of alternative approaches to facilitate engraftment and mitigate GVHD including selective peripheral expansion of regulatory
- T cells.
- The development of gene therapeutic approaches for inherited diseases (www.inherinet.org), spin-off acquired diseases, further preclinical development of hematopoietic and mesenchymal stem cell transplantation using gene marked cells.
- The development of hematopoietic stem cell transplantation using alternative donors and/or alternative stem cell sources.
Theme 3: Malignant transformation of hematopoietic stem cells
Prof. dr. R. Delwel, Prof. dr. I.P. Touw, dr. P. Valk, dr. M. Raaijmakers
The research program aims to elucidate key regulatory abnormalities of leukemogenesis. Emphasis of the program is currently on growth factor receptor and signal transduction derangements and perturbations of transcription and epigenetic control determining functional abnormalities of survival, proliferative, cell cycle, and maturation fates of hematopoietic stem cells.
Another section addresses the role of the interaction between hematopoietic cells and their microenvironment in leukemogenesis. Specific focus is on leukemic progression of leukemia predisposition states, including severe congenital neutropenia (and the role of G-CSF receptor defects (nonsense mutations) in this process) and myelodysplasia.
Genes responsible for leukemic transformation are frequently located near non-random chromosomal translocations. However, in approximately 50% of the clinically diagnosed myeloid leukemias no cytogenetic abnormalities have been detected. Furthermore, in a number of cases that do carry a cytogenetic abnormality the genes located near the breakpoints are still unknown. Moreover, since leukemia is believed to be a multi-step process, aberrant expression of different disease genes affecting multiple pathways are required to obtain full leukemic transformation. An alternative procedure to identify leukemia disease genes is the cloning of common virus integration sites (cVIS). This approach has proven to be a sensitive tool to identify novel proto-oncogenes as well as tumor-suppressor genes. In fact, several genes located near chromosomal breakpoints or otherwise aberrantly expressed in human hematopoietic malignancies have been identified through retroviral insertional mutagenesis in murine leukemias or lymphomas as well, e.g. Evi, Evi2 (NF), Evi6 (Hoxa9), Bcl1 (Cyclin D1), N-Myc, and Erg. The two main lines of investigation that follow from the identification of novel transforming genes in myeloid leukemia are aimed at:
- the mechanisms of myeloid transformation using in vitro and in vivo models
- the role of these mechanisms in human disease.
By means of high throughput sequencing, gene array analysis and real-time PCR we study in a large cohort of AML (± 300 cases) the involvement of novel ‚”leukaemia disease”genes identified by retroviral insertional mutagenesis. Novel disease genes based on the mouse and primary AML screen and, which predict unique pathways and mechanisms of transformation, have been and will be selected for further study. Inducible in vitro and in vivo models will be applied to unravel the exact mechanism of transformation by the distinct transforming genes that have been or will be identified. A program has been designed to assess the clinical significance (prognostic) of findings from high throughput expression profiling and mutational analyses and implement these in clinical molecular diagnostics, and identify targets for treatment intervention.
Theme 4: Diagnosis, classification and treatment evaluation of leukemias and malignant lymphomas
Prof. dr. J.J.M. van Dongen, dr. A.W. Langerak, dr. F.J.T. Staal, dr. V.H.J. van der Velden
This research program focuses on the diagnosis and classification of leukemias and malignant lymphomas as well as on the evaluation of treatment effectiveness during follow-up via detection of low frequencies of malignant cells, i.e. detection of ‚ “minimal residual disease” (MRD). The research program combines molecular and cellular studies on normal and malignant hematopoiesis, particularly focusing on immature lymphoid differentiation. The various types of lymphoid malignancies (leukemias and lymphomas) resemble their normal counterparts. Despite this comparability, the malignant cells exhibit aberrant cellular and genetic characteristics, which can be used for diagnosis, classification, and MRD studies. Thorough insight into normal lymphoid differentiation appears to be highly relevant for translation of new immunobiological information into improved diagnostics. The research program consists of three main projects:
Normal and aberrant V(D)J recombination in leukemias and malignant lymphomas: basic aspects and diagnostic applications
V(D)J recombination of immunoglobulin (Ig) and T-cell receptor (TCR) genes is a key process during early lymphoid differentiation, which is required to establish a broad repertoire of antigen-recognizing receptors. Although the V(D)J recombination process is tightly regulated, aberrant V(D)J recombination occurs, resulting in the coupling of Ig/TCR loci to oncogenes. As a consequence, the involved oncogene is transcriptionally deregulated, eventually resulting in a block in lymphoid differentiation. This differentiation arrest is postulated to lead to a pre-leukemic cell population. Multiple additional genetic hits will result in overt (acute) leukemias or lymphomas.
Insight into normal and oncogenic recombination events will shed light on the pathogenic mechanisms underlying acute leukemia formation. This fundamental knowledge can be translated into better prognostic classification and improved treatment stratification of lymphoid malignancies. As a direct spin-off, these studies might contribute to the identification of novel therapeutic targets.
Immunobiology of acute leukemia and treatment evaluation
Acute leukemia is the most common form of cancer in childhood. Current treatment protocols, consisting of chemotherapy with or without stem cell transplantation can cure the vast majority of patients. However, in 20 to 40% of children the leukemia sooner or later reappears. Apparently, low numbers of leukemic cells, that is ‚”minimal residual disease” (MRD), remain present despite the therapy and finally result in a relapse. How can we detect these low levels of leukemic cells and how can we use MRD information for improving clinical outcome? Over the last couple of years we have developed PCR methods that can detect one leukemic cell amongst up to one million normal cells. Our studies in children with acute lymphoblastic leukemia (ALL) show that such detection of MRD is a very powerful and independent prognostic factor that allows the recognition of patients at high or low risk of relapse.
Our current studies are focused on the development of other sensitive methods for MRD detection, particularly flow cytometric immunophenotyping, and on the evaluation of the clinical significance of MRD in children with acute myeloid leukemia, infants with ALL, and in specific genetic subgroups of childhood ALL. The aims of these studies are to improve MRD monitoring and to establish its clinical significance, thereby allowing patient-tailored therapy of children with leukemia. Such patient-tailored therapy will hopefully result in an improved clinical outcome in children at high risk for relapse and in less intense therapy, and thereby less side effects, in children with a very low risk of relapse.
Gene expression profiles in immature lymphoid cells and acute lymphoblastic leukemias
Gene expression profiles determine the differentiation lineage, developmental stage, and activation stage of the involved cells. Just like in any other cell type, regulation of gene expression in lymphocytes is largely controlled at the level of transcription initiation by transcription factors and transcriptional repressors. The study focuses on transcription factors and signaling routes that are controlling the most immature steps of lymphoid differentiation. In parallel, the abnormal regulation of gene expression in acute lymphoblastic leukemias is studied and compared to corresponding normal immature T and B cell subpopulations. Results of these comparative studies are being exploited for developing new diagnostic tools.
Theme 5: Implementation of molecular diagnostics and novel therapeutic strategies into clinical practice
Prof. dr. J.J. Cornelissen, Prof. dr. R. Delwel, Prof. dr. B. Löwenberg, dr. P. Lugtenburg, Prof. dr. P. Sonneveld, dr. P. Valk, dr. J.M. Zijlmans
Within this theme we link the identification of the molecular mechanisms in the development of hematopoietic neoplasm (in retroviral models and high throughput analysis of clinical samples) to developmental diagnostics and therapeutics and we evaluate and implement clinical investigational procedures. Key-issues of this theme are:
- Clinical trials and correlative lab studies
- Prognostic factors and clinical decisions
- Impact of genetic studies on diagnosis and treatment
- Molecular therapeutics (e.g., ATRA treatment of APL, use of imatinib in CML)
- Ethical issues in clinical trials
Early implementation of potential active oncolytic agents (small molecules) in a controlled clinical trial setting of Phase I/II trials, designed for designated targets in hemato-oncology diseases:
A Clinical Trial Unit (CTU) for this specific goal is operative. Through this unit we have been able to get access to promising ”pipe-line” products from international development programs of several pharmaceutical companies to test in our programs for these diseases. The department has established a leading role in initiating and conducting pivotal clinical studies with new agents in local phase I/II trials and national phase II/III clinical trials with targeted therapies, including Imatinib in Chronic Myeloid Leukemia, Bortezomib in Multiple Myeloma, Gentuzumab and farnesyl Transferase Inhibitors in Acute Myeloid Leukemia and Histone Deacetylase Inhibitors in various leukemias.
Development of allogeneic stem cell transplantation into a widely applicable modality of immunotherapy of leukemia, lymphoma and related diseases:
This program has been built on a 20-year experience of allogeneic and autologous stem cell transplantation. It has transformed from an experimental treatment modality into a well-structured program that focuses on ‚”graft vs leukemia‚” as a means to control and eradicate Minimal Residual Disease (MRD).
The program has developed special interest and background in
- immune reconstitution after stem cell transplantation;
- broad application of ‚”Reduced Intensity Conditioning“as a non-toxic approach to immunotherapy;
- Cellular immunotherapy of MRD by Reduced Intensity Conditioning (RIC). This program has been extended into a broad approach to the value of RIC in prospective trials in leukemia and myeloma. A part of the program dealing with viral activation and immune reconstitution is conducted in collaboration with the department of Virology (Prof. A. Osterhaus).
National and international conducted phase III trials on critical questions in hemato-oncology diseases:
The department of Hematology has an initiating and leading position in (inter-)national trials groups such as the national trial group HOVON and the European Organization for Research and Treatment of Cancer (EORTC). The number of patients included in these trials exceeds 10.000. Many trials have been conducted together with parallel biological studies on tumor samples. In addition, members of the scientific staff are coordinators of clinical trials that are conducted in the EORTC. The activities in this field reflect the focus on translational medicine, which has been defined as the most important challenge for clinical research in the department. An extensive data and tissue bank has been generated. This source is now being used for large-scale genomic analysis for disease-related risk analysis, based on high-throughput techniques.