Immune Pathophysiology of Aplastic Anemia and Immunosuppressive Treatments

National Heart, Lung, And Blood Institute

Investigators

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Abstract

In aplastic anemia, the bone marrow is replaced by fat, and very low peripheral blood counts leading to 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 autoimmune and immunodeficiency syndromes. 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 most treated patients, and immunosuppressive therapies (IST) are the most frequent treatment of marrow failure worldwide. 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 global 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 ancillary research laboratory samples, 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 from the original publication. Relapse is frequent but usually amenable to oral therapies and without impact on survival. Patients who have survived at least 2 years post-IST have normal prospects of survival. Prediction of the development of MDS and AML, the most serious late complications of aplastic anemia, has been a major focus. Age remains the major risk factor, but additionally larger datasets have allowed recognition of patterns of clonal hematopoiesis of significance. The presence of ASXL1- (but not DNMT3A-) and RUNX-mutated clones significantly increases the likelihood of evolution. Sample collection for single cell work in the extension protocol already has yielded striking results, using the advanced techniques of single cell RNA sequencing (scRNAseq) and time-of-flight flow cytometry (CYToF). Cytotoxic lymphocytes in marrow are increased in AA and markedly reduced by triple therapy (but not eliminated); single cell techniques based on sequencing and flow cytometry are concordant in identification of unsuspected subsets of CD8 cells that are distinct to marrow failure and vary with treatment. Conversely, stem cells are absent on presentation and increase in responding cases. T cell receptor usage, aberrant signaling pathways and clone size changes are concordant with our data in large granular lymphocytic leukemia. In a second, novel clinical protocol for treatment-naive disease, low risk oral therapy, cyclosporine and eltrombobag, is initiated before admission to the Clinical Center, for immunosuppression to prevent stem cell loss to lymphocyte cytotoxicity and for stem cell stimulation early. Telemedicine proved especially useful during the pandemic. 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. Also related to the pandemic, we have observed marrow failure following on both viral infection and vaccination, both amenable to IST; fortunately, patients appear to be protected by vaccination, even when immunosuppressed with cyclosporine, and mount normal humoral and cellular responses to viral antigens. 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. Following on our studies of innate immune responses, which disclosed roles for macrophage production of tumor necrosis factor and for myeloid suppressor cells, we have examined megakaryocytes with immune functions. In model systems in which irradiation is omitted, we observed persistence of megakaryocytes morphologically in animals, with cell surface and transcriptional features of immune megakaryocytes. In preliminary co-culture experiments, immune megakaryocytes, even at very low target to effector cell ratios, decreased hematopoietic colony formation in vitro. We have extensively tested the therapeutic efficacy of a commercial Jak1/Jak2 inhibitor, ruxolitinib, in our murine models. 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 other immunosuppressive agents such as murine ATG and cyclosporine. Survival is excellent after brief exposure to drug with essentially normal blood counts. Long term follow-up of animals after recovery from marrow failure and in healthy controls exposed for months to ruxolitinib show minimal deficiencies of hematopoiesis in progenitor and stem cell assays and only the expected lymphocytopenia. We also tested ruxolitinib in combination with cyclosporine, both at suboptimal efficacy doses, and the drugs administered simultaneously are tolerated without toxicity and similarly efficacious compared to full doses of either administered alone. Other more specific jakinibs have not been as effective as ruxolitinib in our model, but testing of more agents is in progress. Our results support a clinical trial of ruxolitinib in a variety of immune marrow diseases, and a protocol to utilize escalating drug doses has received FDA and IRB approval, and this expensive drug will be provided by Incyte and Novartis, its manufacturers. We anticipate our umbrella protocol beginning accrual in fall, 2023, for patients with relapsed and refractory aplastic anemia, moderate aplastic anemia, low risk myelodysplastic syndromes, pure red cell aplasia, large granular lymphocytic leukemia. If there are signals of efficacy with tolerable toxicity, the studies will be extended in each disease as appropriate, and ultimately to treatment-nave severe aplastic anemia as replacement for ATG, avoiding hospitalization and infusion risks from this incompletely defined biologic agent.