In aplastic anemia, the bone marrow is replaced by fat, and peripheral blood 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 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. In clinical work, three protocols are actively accruing patients; several others are in the data analysis phase. In the flagship study of eltrombopag, an oral thrombopoietin mimetic, added to standard immunosuppression, we currently have over 150 patients on study (sample size was 93 for the original study, reported in the New England Journal of Medicine; the extension phase that utilizes the regimen in the third cohort is intended to add a further 87 patients). Overall and complete response rates are consistent with the initial report, and survival very high. Evolution to myelodysplastic syndrome and acute myeloid leukemia, the most serious late consequent of acquired aplastic anemia, appears to be equivalent or lower than historical results. The major value of this study will be defining late risks as well as correlates of neoplastic evolution, especially somatic mutations in MDS/AML genes and telomere length and attrition rates. As an adjunct to this protocol, pharmacokinetics are also performed at the end of hospitalization, for eltrombopag blood levels in adults and children. In other related laboratory studies, we have shown that eltrombopag functions as an iron chelator, and also that thrombopoietin levels remain elevated, in contrast to other hematopoietic growth factors, for many months and years, even in responding patients. In a second protocol, we are assessing rapamycin (sirolimus) as an agent to induce tolerance and prevent relapse, based on animal models described below. Patients who have hematologically responded to immunosuppressive therapy and per protocol or electively discontinue cyclosporine are randomized to 3 months of sirolimus versus no therapy and followed for two years, with relapse the primary end point. With accrual to about 1/3 of the total 108 planned, study drug has been well tolerated. There are signalsrelapses, deaths, transfusions, and platelet countsthat suggest a difference between the study arms. Our third active study is to determine the clinical benefit and effect on telomere attrition of two low doses of danazol, a synthetic androgenic steroid. The rationale for this protocol is our demonstration that high dose danazol improved blood counts and possibly also stabilized extramedullary disease in patients with constitutional telomeropathies; they also surprisingly showed telomere elongation. In the current protocol, the dose has been reduced 50% or 75%, the duration of treatment is briefer, the inclusion criteria have been broadened, and several methods of telomere length determination are employed. In the research laboratory, our mouse model of immune marrow failure has yielded interesting data. In a systematic comparison of rapamycin to cyclosporine, we showed benefit to both in preventing disease but marked differences in the cytokine profile, effects on cytotoxic effector T cell clones, and T regulatory cells. These results comprised part of the rationale for our clinical protocol. Additionally, rapamycin has direct effects on stem cells, not at steady state but under conditions of hematopoietic stress: after alkylating agents and metabolic poisons, while blood counts do not differ, rapamycin doubled hematopoietic stem cells, as measured phenotypically and functionally. In other experiments involving this model, we attempted to expand on our published work that showed interferon-gamma was critical in inducing T cell mediated marrow failure, testing instead another type I cytokine, tumor necrosis factor-alpha. Surprisingly, TNF appeared dispensable in donor lymphocytes but was required in host animals. TNF of host macrophage origin appeared to license donor T cells to produce IFN; in human experiments, macrophages from patients showed intracellular TNF, and TNF augmented IFN production in activated cytotoxic T cells from healthy donors. In contrast to IFN and TNF, PDF deficiency augmented immune marrow failure when minor antigens were histo-incompatible. We have also explored the role of the microbiota in hematopoiesis in animals, confirming that animals raised conventionally show marked skewing of CD8 T cell heterogeneity, and showing that animals raised in dirty environments have mildly enhanced stem cell numbers and functionality. In translational experiments utilizing patient samples, we employed deep sequencing and high resolution flow cytometry to define expanded populations of effector memory CD+ T cells in patients with aplastic anemia, including demonstration of homology in antigen-binding CDR3 regions among patients. We extended previous work on circulating exosomal microRNAs in marrow failure syndromes. Having published our analysis of single cell RNA transcription in normal and aneuploid human CD34 bone marrow cells, we have re-analyzed these data focusing on long non-coding RNAs, important regulatory elements not yet well characterized in hematopoiesis. We discovered much greater heterogeneity among individual cells in LNCRNA compared to mRNA expression, allowing for better definition of stem versus progenitor cells, lineage, and maturation. Single cell RNAseq is now broadly applied in the laboratory, with analyses ongoing of lymphocytes in large granular lymphocytosis, in constitutional marrow failure syndromes, and to detect loss of heterozygosity, as for example in the region of chromosome 6 that encodes HLA.In collaborative work, we established a method for detection of somatic mutations in human cell free plasma, which will be a platform for planned studies of banked specimens from patients with a variety of inflammatory diseases.
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