In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood counts - - of white blood cells, red blood cells, and platelets - - fall to extremely low levels, leading to death from anemia, bleeding or infection. Aplastic anemia is a disease of young persons and in its severe form is almost invariably fatal untreated. Historically, aplastic anemia has been linked to chemical exposures, in particular benzene; it is an idiosyncratic complication of some medical drug use; it occurs as a rare event in pregnancy and following seronegative hepatitis; and the diseases associated with certain immunologic conditions. The chance observation that some patients post-bone marrow transplant recovered their own marrow function led to the inference that the immunosuppressive conditioning regimen might have treated an underlying immune-mediated pathophysiology. Purposeful administration of antithymocyte globulin (ATG) has led to hematologic recovery in the majority of treated patients. Laboratory data have also revealed abnormalities of the immune system: lymphocyte populations that induce apoptosis in hematopoietic target cells by the Fas-mediated pathway, and oligoclones of effector T cells which express type 1 cytokines, especially gamma-interferon. The Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. Clinical protocols in the Branch have centered on utilization of the synthetic thrombopoietin mimetic eltrombopag in bone marrow failure. First, we have continued accruing patients to our extension trial of eltrombopag as a single agent in patients with refractory disease; the response rate of 40-50% has been maintained and the rate of clonal evolution to myelodysplastic syndrome and acute myeloid leukemia has not increased. Genomics are being employed in order to determine whether mutations in candidate genes are responsible for untoward late hematologic effects. Eltrombopag is also being employed as a single agent in patients with low-risk myelodysplastic syndrome and in moderate aplastic anemia or single lineage bone marrow failure, such as pure red cell aplasia. Responses have been seen in all categories, in some cases approaching 50% hematologic recovery. However, as yet, there is not clear evidence that MDS in its usual presentation in the elderly with cytopenias has a high response rate to eltrombopag; MDS following aplastic anemia has responded to this therapy. Our major clinical research protocol is the combination of conventional immunosuppression (horse antithymocyte globulin and cyclosporine), with eltrombopag in treatment-nave severe aplastic anemia. This protocol has now accrued about 75 patients in three cohorts. In the first cohort of 31 patients, eltrombopag was initiated two weeks after the first dose of ATG and continued for six months. In the second cohort, eltrombopag exposure was limited to the first three months; in the third and current cohort, eltrombopag is administered with ATG treatment and continued for six months. Overall and complete response rates are similar in the three cohorts, with 75-90% of patients responding by three or six months, and with a complete response rate approximately 30%. Both rates are much higher than observed historically in patients who received ATG and cyclosporine (approximately 65% and 10%, respectively). The rate of clonal evolution appears to be lower than in patients with refractory disease, although some cases of evolution to monosomy 7 requiring stem cell transplant intervention have occurred. Long-term survival data will be accrued, as will genomic analysis of patients at landmark time points to monitor for somatically mutated clones, especially in patients who evolved to MDS/AML. Negotiations are underway for continuation of further trials of eltrombopag in hematologic disease at the Clinical Center, under a Collaborative Research and Development Agreement, now with Novartis. In the basic research laboratory, we are interested in the mechanism of action of eltrombopag in aplastic anemia, as patients have high endogenous thrombopoietin levels; erythropoietin and myeloid colony stimulating factors are ineffective in patients with bone marrow failure, presumably because patients are already producing large quantities of hematopoietic growth factors in physiologic response to cytopenias. One curious observation is that thrombopoietin levels remain elevated in patients post-immunosuppressive therapy for long periods of time, even with complete blood count recovery. We hypthothesize that thrombopoietin is regulated by at least in part by megakaryocyte mass, which may not return to normal in aplastic anemia even when platelet counts are adequate. In collaboration with Andre LaRochelle, a tenure-track scientist, and with Cynthia Dunbar we also participate in mechanistic experiments utilizing primary cells and hemapoietic cell lines. Following on work of others, we are investigating whether eltrombopag can circumvent a block in hemapoietic growth factor signal transduction in the presence of inflammatory cytokines such as gamma-interferon. In animal models, with which our laboratory has had extensive experience, we have completed investigations of the potentially dual biologic roles of gamma interferon. Others have claimed that gamma interferon increases hematopoietic stem cell number and drives myelopoiesis. However, in our studies, gamma interferon increases phenotypically defined hematopoietic stem cells, but these cells have poor functional ability; as in transplant experiments. Likely, Sca-1 upregulation on stem cells is responsible for this discrepancy. Conversely, gamma interferon induces Fas expression and apoptosis, especially in the presence of Fas ligand or activated cytotoxic effector cells. Our recent data are therefore consistent with historical results in which gamma interferon has been shown to negatively regulate hematopoiesis, compatible with its role in immune-mediated bone marrow failure in humans. Finally, because relapse remains a serious problem in patients with aplastic anemia who have been treated with immunosuppressive therapy, we have readdressed the role of a specific immunomodulatory drug, rapamycin or sirolimus, in our mouse model of immune-mediated bone marrow failure. These experiments are also inspired by the successful utilization of sirolimus in the setting of high risk stem cell transplants in sickle cell disease, as performed by our colleague John Tisdale in NHLBI. In animal experiments, rapamycin is very effective in preventing stem cell destruction in the immune-mediated aplastic anemia mouse model. We have determined optimal dose and regimen. Further, there are marked differences in lymphocytes as determined by flow cytometry phenotyping of the bone marrow of affected animals, and in transcriptional profiling. Current experiments investigate the role of T-regulatory cells and direct induction of apoptosis of effector cells in this animal model. These animal data, as well as theoretical considerations, should lead to a protocol in which rapamycin is substituted for cyclosporine in an effort to induce tolerance in aplastic anemia patients after hematologic recovery.