Immune Pathophysiology of Aplastic Anemia and Immunosuppressive Treatments

National Heart, Lung, And Blood Institute

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Abstract

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 mainly young persons and when severe is almost invariably fatal when untreated. 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 with immune system diseases, both autoimmunity and immunodefiency states. The serendipitous 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. More recently, eltrombopag, a thrombopoietin mimetic, was shown by us to be effective in improving clinical outcomes; data from our Branch were the basis for approval by the FDA for use of this drug in both refractory and treatment-nave severe aplastic anemia. Our section within the Hematology Branch has been a leader in both scientific research and medical studies of aplastic anemia pathophysiology and treatment. In clinical work, our protocol for severe, treatment-nave aplastic anemia was extended to collect more clinical data and subsequently for purposes of ancillary research laboratory studies, both aimed in particular at long-term outcomes. Our large cohort is of unique value in providing accurate estimates of response rates, relapse, and evolution to myeloid malignancies with now standard therapy. Overall and complete response rates are consistent with the initial report, and survival remains very high. There have been no significant changes in reported rates of relapse and malignant evolution. We have used both the triple therapy cohort to identify risk factors for later development of MDS and AML. Age remains the major risk factor, but additionally large datasets have allowed recognition of patterns of clonal hematopoiesis of significance. For one example, ASXL1 (but not DNMT3A) mutated clones increase the likelihood of evolution. Another example is the pattern of HLA and deletions and mutations within the HLA complex that suggest distinct modes of escape of malignant clones from immune surveillance. Sample collection for single cell work in the extension protocol already has yielded striking results. Cytotoxic lymphocytes in marrow are increased in AA and markedly reduced by triple therapy (but not eliminated); conversely, stem cells are absent on presentation and increase in responding patients. RNA sequencing of single cells shows concordant results, and changes in signaling pathways and clone size similar to our just published data in large granular lymphocytic leukemia. Of particular interest, expansion in the hematopoietic compartment occurs mainly among more committed mature stem cells. In a second clinical protocol for treatment-nave disease, we initiate low risk oral therapy, cyclosporine and eltrombobag, in order to introduce immunosuppression and stem cell stimulation early and to prevent loss of hematopoietic cells to T cell killing. Telemedicineespecially useful during the pandemic--has been employed to enroll and follow patients until they are admitted to the Clinical Center for definitive anti-thymocyte globulin infusion (if needed). Unexpected adverse events, especially drug toxicity, errors in diagnosis, and patient compliance to outpatient visits and physician directives have been excellent and the response rate as high or superior to the standard regimen. Our relapse prevention trial, assessing rapamycin (sirolimus) as a lymphocytotoxic tolerogenic agent continues to accrue. Study drug has been well tolerated. Our retrospective data indicate that future protocols, preferably multicenter, should test sirolimus at an earlier time point, around six months, when the cyclosporine dose is sharply reduced and eltrombopag discontinued. Our mouse model of immune marrow failure has continued to yield interesting and important data. Based on our finding of a role of macrophages and tumor necrosis factor in the pathologic immune response, experiments have been performed to test other components of the innate immune system. As reported previously, Toll receptors and a variety of cytokines do not appear to play roles in this model. However, myeloid suppressor cells are active in reducing the T cell response and ameliorating hematopoietic failure, Cell therapy has been used in graft-versus-host disease and may have utility in refractory immune marrow failure. We have extensively tested the therapeutic efficacy of a commercial Jak1/Jak2 inhibitor, ruxolitinib, in our murine models. This drug is ingested as a component of chow to mice. Ruxolitinib not only completely prevents marrow failure when administered prior to infusion of lymph node cells but is effective even days after infusion, in contrast to our previous experience with a variety of immunosuppressive agents. Survival is excellent after brief periods of exposure to drug with essentially normal blood counts and marrow cellularity long term, and only modest evidence of residual stem cell damage. Plasma cytokines remain normal and the expected signaling pathways are blocked by this jakinib. Drug toxicity in both treated animals in the model and in healthy animals is minimal. These results support a clinical trial of ruxolitinib in a variety of immune marrow diseases, relapsed and refractory AA as well as low risk MDS and pure red cell aplasia, and potentially as replacement for ATG, avoiding hospitalization and infusion risks from this incompletely defined biologic agent.