The overall objective of this proposal is to apply molecular biological analysis of clonal growth and growth factor expression in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). These disorders are believed to result from genetic alterations in hematopoietic stem cells leading to a block in normal differentiation and autonomous growth of immature myeloid cells. Markers capable of identifying non-dividing mature cells as progeny of the malignant clone have suggested that the block in differentiation may be incomplete in some cases, both at the time of diagnosis as well as complete remission. Moreover, it has been suggested that the mechanism of autonomous growth of leukemic progenitor cell clones may be related to autocrine secretion of growth factors. The proposed studies will focus on these issues by examination of cells from patients with AML and MDS. Research in our laboratories has identified clonal growth of leukemic cells or their progeny via noting allelic loss of a restriction fragment length polymorphism (RFLP) associated with the X-linked phosphoglycerate kinase (PGK) gene. Other work has documented the presence of mutations in codons 12 or 13 of the K- or N-ras protooncogene sequences in diagnostic samples by polymerase-chain reaction-based sequencing. Growth factor expression has been demonstrated at both the single cell level by in situ hybridization and at the clonal level by amplification of cDNA obtained from reverse transcription of specific mRNA sequences (reverse PCR). These techniques provide the basis for examination of samples from patients with AML and MDS at diagnosis, after therapy, and during remission. The proposed work will study clonal growth in cells from patients with AML and MDS by identification of loss of RFLPs in the PGK gene, somatic mutations in ras protooncogenes and rearrangements of satellite DNA bands (Specific Aim 1). In order to determine the relevance of growth factor expression and response in individual cells and leukemic clones we will determine expression of GM-CSF, M-CSF, and IL-3 mRNA by both reverse PCR and in situ hybridization (Specific Aim 2) and monitor protein phosphorylation with anti-phosphotyrosine antibodies (Specific Aim 3). Finally, we will correlate the results of our proposed laboratory studies with the clinical features (FAB subtype, remission status, treatment response) of the patients from whom the samples were obtained (Specific Aim 4). These studies should provide insights into the importance of both clonal growth and growth factor dependence at various stages of the progression of AML and MDS. Such information could lead to improved prognostic data as well as more effectively timed therapeutic interventions.