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. Recent clinical protocols in the Branch have centered on utilization of the synthetic thrombopoietin mimetic eltrombopag in bone marrow failure. 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. Eltrombopag is also being employed as a single agent in patients with low risk myelodysplastic syndrome and in moderate aplastic anemia and single lineage bone marrow failure, such as pure red cell aplasia. Our major clinical research protocol is the combination of conventional immunosuppression, with eltrombopag in treatment-naive severe aplastic anemia. The protocol has now completed accrual and all accrued patients have been followed to the primary end point at six months. The overall response rate at three months is 80% and at six months 87%, and we have met our goal of at least doubling the complete response rate, with CRs at three months at 30% and six months at 40%. Of note, in cohort 3, in which eltrombopag is begun on day 0 and continued for a full six months, the six month overall response rate is 94% and the complete response rate 61%. These results are far superior to those obtained with immunosuppression alone. Evolution has occurred to myelodysplastic syndrome and acute myeloid leukemia, usually manifested as the appearance of monsomy 7 on bone marrow cytogenetics, but the rate appears comparable to date with immunosuppression alone. Severe cutaneous eruptions have been the major toxicity. Eltrombopag has not caused untoward hepatotoxicity, even with concomitant use of other hepatotoxic drugs and biologics. The overall prevalence of somatic mutations is as we have reported with immunosuppression alone and BCOR, and these mutations have been associated by us with favorable outcomes. Of note, germline RTEL1 mutations were present in two patients who later underwent clonal evolution. In the basic laboratory, effort has been made to respond to and refute two prominently published papers. In one, adipocytes were suggested to be suppressors of hemopoiesis, based on studies in the mouse utilizing a putatively specific inhibitor of PPAR. However, we showed that there was poor correlation between fat and hematopoietic cell suppression and that the PPAR antagonist influenced the immune response and functioned best in settings of immune rejection or immune suppression of hematopoietic cells and hematopoiesis. A second publication suggested that IFN- increased murine hemapoietic stem cells. In our new work, we showed that IFN- expanded bone marrow KSL cell number but reduced their function, as these cells engrafted poorly in a competitive repopulation model. We confirmed our earlier work that IFN- was toxic to hemapoietic cells, acting through FasFas ligand to induce apoptosis and to activate cytotoxic T cells. In collaboration with colleagues at Kings College Hospital in the United Kingdom, we performed deep phenotyping of T-regulatory cells of patients, showing that specific T-regs, identified by time of flight flow cytometry, were present in aplastic anemia and predicted response to ATG and cyclosporine. In paroxysmal nocturnal hemoglobinuria, we utilized RNA-seq as an unbiased method for detection of transcripts, and found that granulocytes in these patients expressed a unique deficiency that implicates a novel TNF pathway in immune activation. We showed that a novel microRNA signature from T cells is linked to acquired aplastic anemia and may be a biomarker to distinguish AA from MDS and to predict response to therapy. We have introduced two new methodologies into the laboratory: CRISPR/Cas9-mediated gene editing and single cell RNA-seq. We employed cell lines to mutate or knock out genes implicated in clonal evolution in aplastic and in predisposition to myeloid neoplasms. For ASXL1, knock out clones of U937 leukemia cells showed few differences in cell morphology, proliferation, cell cycle and susceptibility to apoptosis. However, when monocyte/microphage differentiation was induced, ASXL1 knock out clones showed a marked deficit in differentiation, and RNA-seq implicated pathways related to cell death and survival, including TNF signaling. For DNMT3A, gene editing of K562 leukemia cells also had no consistent effect on most parameters of proliferation, differentiation and apoptosis. However, DNMT3A clones showed marked chromosome instability, with highly diverse patterns of aneuploidy and translocations. In single cell RNA-seq experiments utilizing human bone marrow CD34 cells; we used computational methods to define pathways of differentiation, and we were able to distinguish primary aneuploid cells, known to have monosomy 7 from diploid cells from the same patient sample. Distinction of chromosome 7 loss allowed characterization of a transcriptome program showing immune response genes to be abnormally expressed in the aneuploid population.

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Zhao, Zhen; Sun, Qian; Sokoll, Lori J et al. (2018) Eltrombopag mobilizes iron in patients with aplastic anemia. Blood 131:2399-2402
Giudice, Valentina; Wu, Zhijie; Kajigaya, Sachiko et al. (2018) Circulating S100A8 and S100A9 protein levels in plasma of patients with acquired aplastic anemia and myelodysplastic syndromes. Cytokine :
Giudice, Valentina; Banaszak, Lauren G; Gutierrez-Rodrigues, Fernanda et al. (2018) Circulating exosomal microRNAs in acquired aplastic anemia and myelodysplastic syndromes. Haematologica 103:1150-1159
Dulau-Florea, Alina E; Young, Neal S; Maric, Irina et al. (2018) Bone Marrow as a Source of Cells for Paroxysmal Nocturnal Hemoglobinuria Detection. Am J Clin Pathol :
Zhao, Xin; Feng, Xingmin; Wu, Zhijie et al. (2018) Persistent elevation of plasma thrombopoietin levels after treatment in severe aplastic anemia. Exp Hematol 58:39-43
Albitar, A; Townsley, D; Ma, W et al. (2018) Prevalence of somatic mutations in patients with aplastic anemia using peripheral blood cfDNA as compared with BM. Leukemia 32:227-229
Hollinger, Maile K; Giudice, Valentina; Cummings, Nicole A et al. (2018) PD-1 deficiency augments bone marrow failure in a minor-histocompatibility antigen mismatch lymphocyte infusion model. Exp Hematol 62:17-23
Banaszak, Lauren G; Giudice, Valentina; Zhao, Xin et al. (2018) Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing. Blood Cells Mol Dis 69:10-22
Wu, Zhi-Jie; Zhao, Xin; Banaszak, Lauren G et al. (2018) CRISPR/Cas9-mediated ASXL1 mutations in U937 cells disrupt myeloid differentiation. Int J Oncol 52:1209-1223
Giudice, Valentina; Feng, Xingmin; Lin, Zenghua et al. (2018) Deep sequencing and flow cytometric characterization of expanded effector memory CD8+CD57+ T cells frequently reveals T-cell receptor V? oligoclonality and CDR3 homology in acquired aplastic anemia. Haematologica 103:759-769

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