-Dr. Melvin Freedman, SCNIR, "Neutropenia Support Assoc., Inc., Volume 9 Winter 1997"

Patients with congenital bone marrow failure have defective hematopoiesis, which is evident early in life. The faulty marrow function varies from single lineage cytopenia to full-blown trilineage aplastic anemia. Since growth and differentiation of bone marrow progenitor cells are controlled by stage and lineage specific growth factors, we hypothesize that in a proportion of patients with congenital bone marrow failure disorders, the defect in hematopoiesis is due to a mutation in a gene coding for either a specific growth factor or its receptor. According to this hypothesis, a defect in a gene coding for a growth factor receptor will manifest itself as unresponsiveness of hematopoietic progenitor cells to that particular growth factor. In contrast, a mutation in a gene coding for a growth factor produced by T-cells or stromal cells may result in an inability of bone marrow stroma or T-cells to sustain hematopoietic colonies in vitro. In this way an assessment of the effects of growth factors on hematopoietic colony growth will provide an initial indication of the locus of a mutation in a particular patient. We propose to test this hypothesis initially by determination of the expression (by internally controlled PCR and flow cytometry), and function (by clonogenic assays) of hematopoietic growth factors and their receptors in cells from patients with bone marrow failure. The results from these experiments will provide the basis for a more direct determination of the expression of a particular growth factor or its receptor followed by the identification of a structural mutation in the specific gene. The following specific aims will be addressed.

  1. To assess the ability of bone marrow cells from patients with congenital bone marrow failure to form hematopoietic colonies and to respond to growth factors. To assess the ability of long term stromal cell cultures from patients with congenital bone marrow failure to support normal hematopoiesis in vitro. To compare the expression of growth factors receptors on bone marrow cells from normal subjects and from patients with congenital bone marrow failure. And to compare the expression of growth factors by cultured bone marrow stromal cells and by activated T lymphocytes from normal subjects and from patients with congenital bone marrow failure.
  2. To identify mutations in candidate genes coding for growth factors or their receptors in selected patients with bone marrow failure. Within the classification of the congenital bone marrow failure disorders, there is a category called severe congenital Neutropenia. Because of severe impairment in neutrophil production, these patients develop life-threatening infections. Although the molecular explanation for congenital Neutropenia is not yet known, recombinant granulocyte-colony stimulating factor (G-CSF) overrides the defect when administered clinically and reverses the Neutropenia in patients. In the era prior to G-CSF therapy, there were 2 published cases of congenital Neutropenia evolving into acute myeloid leukemia. Since the widespread use of G-CSF for congenital Neutropenia in the past 5 years, there has been an alarming increase in patients with myelodysplastic syndrome and acute myeloid leukemia. Many of these patients manifest ras oncogene mutations and monosomy 7 in bone marrow cells, and a few have a mutated G-CSF receptor (G-CSF-R). We hypothesize, therefore, that congenital Neutropenia is associated with a genetic predisposition to myelodysplasia/acute myeloid leukemia and that a subgroup can be identified during its multistep pathogenesis by demonstrating specific genetic aberrations. We also hypothesize that G-CSF therapy accelerates the evolution of myelodysplasia/acute myeloid leukemia.

Thus, the specific aims are

  1. To study by routine cytogenetics and by florescent in situ hybridization patients’cells prior to and during G-CSF therapy with regard to loss of pan or all of chromosome 7. To also look for cytogenetically undetectable lesions on chromosome 7 by analyzing sequence polmorphisms by PCR. To detect ras mutations pre and during G-CSF therapy by SSCP, by allele specific restriction enzyme digests, and by cloning and sequencing mutant ras fragments. And to sequence the cytoplasmic domain of the G-CSF-R in marrow cells at diagnosis, during GCSF therapy and at presentation of leukemia.
  2. To use an in vivo SCID mouse model to test the potential of G-CSF at inducing genetic aberrations in patients’ marrow or for selecting genetically unstable marrow cells with these aberrations (ras mutations, monosomy 7, G-CSF-R mutations). Also, in collaboration with others, to identify the signaling partners for the distal differentiation domain of the G-CSF-R in patients, and to analyze the G-CSF/ras pathway in patients’ granulocytes (induced to mature with G-CSF) and in normals.

These studies are certain to provide new and important information. Identification of the molecular defects leading to bone marrow failure will lead to a better understanding of the role of hematopoietic growth factors in normals and in bone marrow failure. In the subgroup of patients with congenital Neutropenia, there is growing concern about malignant transformation while receiving G-CSF therapy. This is the first proposal to address this issue in a comprehensive manner.