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 anti-thymocyte 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. Our section in the Hematology Branch has been a leader in both the scientific and medical studies of aplastic anemia pathophysiology and treatment. In clinical work, our protocol for severe, treatment-nave aplastic anemia continues to accrue patients. The combination of standard immunosuppression with eltrombopag is now standard-of-care for this disease due to FDA approval based on NIH studies. Our cohort of patients, now nearly 200 in total, is of unique value as providing accurate short- and long-term results, particularly response rates, relapse, and evolution to myeloid malignancies with this novel approach. Overall and complete response rates are consistent with the initial report, and survival remains 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: high risk cytogenetic transformation (monosomy 7 and complex abnormalities) at a cumulative incidence of <5% at >2 years follow-up from treatment. We have observed occasional very late evolution to morphologic MDS/AML. Detailed analyses are ongoing, but preliminary data suggest that eltrombopag addition provides less benefit to children than for adults, not explicable by different pharmacokinetics., For monosomy 7, its occurrence bodes a poor prognosis with supportive care and better outcomes with early stem cell transplantation. In a second clinical protocol, we are assessing rapamycin (sirolimus) as an agent to induce tolerance and prevent relapse, based on preclinical data from 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 1/2 of the total 108 planned, study drug has been well tolerated without serious or unanticipated adverse events. There are early signals from the numbers of relapses, deaths, transfusions, and platelet counts that of a difference between the study arms. Our third active study addresses danazol in telomere disease and is described in that annual report. In the research laboratory, our mouse model of immune marrow failure has continued to yield interesting data. We seek to develop a reliable model of chronic aplastic anemia after lymphocyte infusion. Based on evidence that was described in last years report of a role of macrophages and tumor necrosis factor in the pathologic immune response, experiments have been designed to test several toll-like receptors (2,3, and 4) and IL-6 in the model. We have also detected clonal hematopoiesis in mice that were subjected to radiation and alkylating agents, providing a potential model system to study clonal hematopoiesis of indeterminate potential (CHIP) in animals. In translational experiments utilizing patient samples, we have emphasized RNA sequencing in single cells (scRNAseq). Previously, we published our analyses of scRNAseq in normal and aneuploid human CD34 bone marrow cells. The original work was extended to long non-coding RNAs and more recently to the pattern of normal hematopoietic cell differentiation and maturation. We now have completed analyses in the constitutional marrow failure syndrome GATA2 deficiency and in the acquired immune marrow failure disease large granular lymphocytosis. For GATA2, we have identified a signature of deficient hematopoietic differentiation and maturation, likely independent of aneuploid evolution. For LGL, scRNAseq confirmed by sequencing alone the characteristic mono- and oligoclonality of cytotoxic lymphocytes; successful treatment with alemtuzumab, a monoclonal antibody which we have shown to be effective in refractory disease, did not alter T cell clonality or cell number but markedly affected lymphocyte activation. In other work, we have examined loss of HLA expression in hematopoietic cells of patients with aplastic anemia; loss of heterozygosity of the 6p chromosome and somatic mutations of HLC class I genes are frequent in this disease, likely as an escape mechanism from immune attack on marrow target cells; we have found mutations to be equivalently prevalent for multiple different HLA-A and -B alleles, but some alleles may be preferentially affected. In contrast to a published report, HLA gene mutations and specific HLA alleles were not associated with clinical outcomes, particularly clonal evolution. Our Finnish collaborators have characterized private T cell receptor clonotypes that show sequence similarity and demonstrated fluctuations in effector clones with immunosuppressive therapy; they will also seek acquired mutations in immune gene pathways in these cells. In exploration of biologic correlates for our relapse prevention protocol of rapamycin, we have identified a putative signature of relapse based on detailed analyses of circulating cytokines and lymphocyte immunophenotype of banked specimens.
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