Background: Chronic idiopathic myelofibrosis (IM), which is thought to originate at the level of the multipotent hematopoietic stem cell (HSC), is characterized by hyperplasia of morphologically abnormal megakaryocytes and clonal populations of monocytes, shown to release fibrogenic growth factors leading to bone marrow (BM) fibrosis.
IM is thought to be characterized by malignant HSC possessing an extreme proliferative capacity, a unique differentiation program and the potential for self-renewal. Since these characteristics have not been directly documented to date, the comparison of normal and IM HSC provides a starting point for identifying the cell or cells in the normal hematopoietic hierarchy from which IM originates. Although diagnostic criteria of IM have been developed, the cellular and molecular events occurring during the clinical course of IM remain unknown.
Hypothesis: To address these questions, we hypothesize that IM originates at the level of the HSC and that disease progression is due to an increasing burden of malignant HSC and/or changes in their function.
Specific Aims: The specific aims outlined in this proposal include: (1) phenotyping and quantitating the number of CD34+ cells in the peripheral blood (PB) and BM of patients with IM; (2) performing fluorescence in situ hybridization (FISH) and clonality assays to determine if myeloid, lymphoid and endothelial cells in IM are derived from a malignant clone; (3) determining if IM originates at the level of an adult hemangioblast; (4) determining whether the secretion of growth factors and cytokines by IM CD34+ cells, myeloblasts, monocytes, erythroblasts, and megakaryoblasts leads to BM fibrosis, ineffective hematopoiesis, and increased BM microvessel density; (5) determining the NOD-SCID mouse BM repopulating potential of CD34+ cells from the PB of IM patients; and (6) studying IM disease progression in immunodeficient mice.
Study Design: The CD34+ cell population isolated from the PB of patients in various phases of IM will be phenotypically characterized to determine if these cell populations are composed of a different proportion of primitive and differentiated HPC than that present in the control groups. Purified cell populations will be isolated from the PB of female IM patients possessing numerical or structural cytogenetic abnormalities, and FISH analysis and clonality assays will be performed to determine if cells belonging to these different hematopoietic lineages are derived from the malignant IM clone. We also will test the hypothesis that the new blood vessel formation in IM BM is due to the recruitment of endothelial progenitor cells derived from a malignant precursor capable of producing both hematopoietic and endothelial cells. Hematopoietic colonies and cobblestone areas as well as endothelial cells will be cultured from the PB mononuclear cells of female IM patients exhibiting numerical or structural cytogenetic abnormalities and analyzed by FISH analysis and clonality assays to determine if these hematopoietic and endothelial cells belong to the same malignant clone. Chromogenic in situ hybridization (CISH) will also be performed on formalin fixed paraffin embedded sections of BM to further confirm that these chromosomal abnormalities occur in both endothelial and hematopoietic cells.
The role of the autocrine/paracrine production by malignant hematopoietic cells of various cytokines, growth factors, and chemokines in the development of BM fibrosis, ineffective hematopoiesis, and increased BM microvessel density in IM will be analyzed by quantitative PCR for the mRNA expression of these factors on purified populations of both normal and IM CD34+ cells and their differentiated progeny. The secretion of growth factors, cytokines, and chemokines by hematopoietic cells also will be evaluated using ELISA.
To further characterize the IM HSC/HPC, we will transplant either IM PB CD34+ cells or subpopulations of CD34+ cells in limiting dilution into pre-irradiated NOD/SCID mice. Flow cytometric analysis will be performed with monoclonal antibodies to several human antigens to show that the IM HSC are capable of differentiating into multiple hematopoietic lineages over time in the recipient mice. FISH analysis will be performed on the initial CD34+ cells, individually plucked hematopoietic colonies, and cobblestone areas cloned from donor IM patients as well as from human CD45+ cells isolated from the mouse-human chimeras. In addition, when sufficient human engraftment is achieved, CD45+ cells will be isolated and plated in HPC assays, and colonies will be plucked and analyzed by FISH and clonality assays to determine if they are derived from a malignant clone or normal HSC.
Since one of the defining characteristics of an HSC is its ability to self-renew, the ability of human BM cells from the primary NOD/SCID mouse recipient to re-populate secondary NOD/SCID recipients also will be assessed. Utilizing the nude NOD/SCID assay system, we will determine if cells isolated from patients at these various stages of IM have a greater potential to recapitulate the disease, if the sequelae of IM are determined by the characteristics of the IM HSC, and if this potential increases with disease progression.
Relevance: The performance of these studies is critical to gaining understanding of the mechanisms involved in the origins and progression of IM as well as the rational design of new therapeutic strategies.
|Effective start/end date||1/01/04 → 31/12/04|
- U.S. Department of Defense: $507,199.00