The MDS program has contracted due to the death of a principal investigator with a strong interest in this field. Nevertheless, several clinical studies have continued and are nearing completion, and furthermore, the telomere projects (Telomere Diseases project) now substantially overlap with MDS investigations, and some of the data from that work is incorporated in this annual report. In clinical studies, we completed and have published results of a clinical research trial for patients with MDS utilizing a novel suppressor of cyclin D1 termed ON1910.Na. We have previously demonstrated that upregulation of c-myc survivin, and cyclin D1 occurs in CD34 bone marrow cells of patients with aneuploid forms of MDS. Furthermore, knock down of cyclin D1 decreased trisomy 8 cell proliferation and improved hematopoiesis in vitro, suggesting cyclin D1 as a therapeutic target in MDS. Preclinical and clinical studies were performed in patients with high risk as well as trisomy 8 AML who were enrolled in a phase I clinical protocol. ON1910.Na in vitro inhibited cyclin D1 expression and was selectively toxic to trisomy 8 cells. Flow cytometry showed increased mature CD15 myeloid cells and decreased CD34 myeloblasts. In three patients treated with the drug, there were decreased bone marrow blasts and in three patients there was hematologic improvement, in one sustained for almost three years. Hematologic responses were correlated to decreased cyclin D1 expression. These positive preclinical results will be further investigated at other institutions with the sponsorship of Onconova. In other clinical studies, we continue to enroll patients with a high likelihood of response to immunosuppressive therapy - - determined by age, transfusion burden, and histocompatability type - - for therapy with alemtuzumab, a monoclonal antibody broadly reactive with human lymphocytes. A subcutaneous route of administration which allows outpatient delivery in the clinic, has been substituted for intravenous infusions. However, this study may be abbreviated due to unavailability of the drug: alemtuzumab has recently been shown to be effective in multiple sclerosis, and has been formulated by it manufacturer, Pfizer, for this purpose. Under consideration is revision of the current protocol to doses of alemtuzumab that will be used in multiple sclerosis, as an adequate dose has never been determined in vivo in clinical trials. The high response rate that we observed in selected patients suggests that such an approach would be feasible and cost effective, and guidance to the hematology community in the use CAMPATH in MDS and other hematologic syndromes. In the laboratory, we have developed a method based on combining fluorescent in situ hybridization (FISH and flow cytometry) to detect monosomy 7 in interphase cells quickly and utilizing peripheral blood. Monosomy 7 is a serious clonal event in patients with aplastic anemia, and complete or partial deletions of chromosome 7 in general confer poor prognosis in myeloid hematologic malignancies. Therefore, early detection of small clones of monosomy 7 would have great utility, as the current standard of practice is bone marrow examination and conventional cytogenetics or standard FISH. Using peripheral blood samples from patients with MDS known to have a monosomy 7 by conventional cytogenetics, we show that fresh mononuclear cells isolated by sedimentation could be permeabalized, and nucleic acid probes specific to chromosome 7 entered the cell with the cell membrane intact. Healthy donor samples showed distribution of chromosome 7 in a diploid pattern in monocytes and lymphocytes. However, in samples from patients with monosomy 7, a population of monocytes showed distinctly lower fluorescent signal than did accompanying lymphocytes and monocytes, consistent with the myeloid origin of aneuploidy. In patient samples, the proportions of monocytes with monosomy 7 correlated well with monosomy 7 as detected in conventional cytogenetics. Our method can be utilized to monitor patients in our current clinical trials in order to achieve early detection of clonal evolution. Finally, we have investigated in a systematic fashion the origin of MDS in patients with aplastic anemia who evolve, usually to monosomy 7, after immunosuppressive therapy. We have combined both genetic and chromosomal assays to determine which mechanism of oncogenesis dominates. As described in our annual report on telomere diseases, serial samples in patients have shown by single telomere length analysis as well as mean telomere length determination that telomere attrition accelerates prior to or concurrent with evolution. In whole genome sequencing experiments utilizing the same samples, we have detected mutations in pathways related to hematpoiesis, suggesting selection of cells in the stressed marrow failure environment that may offer proliferative advantages. Similarly, some immune pathways also appear affected in sub-populations. However, there has been a noticeable absence mutations in known oncogenes and tumor suppressor genes. If confirmed, these data would suggest that chromosome abnormalities drive the development of MDS and leukemia in this setting. Further experiments are planned with comparative genomic hybridization to detect small deletions and other abnormalities that may occur with critical telomere shortening. Furthermore, in vitro experimental approaches are feasible to determine the role of the chromosome in the absence of genetic lesions in destabilizing the genomes.

Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2012
Total Cost
$1,057,808
Indirect Cost
Name
National Heart, Lung, and Blood Institute
Department
Type
DUNS #
City
State
Country
Zip Code
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