Mixed Chimerism Following Nonmyeloablative Stem Cell Transplantation for Sickle Cell Disease Prevents Intravascular Hemolysis and Restores Endothelial Function.

Abstract Recent studies have highlighted the impact of chronic intravascular hemolysis on endothelial function and end-organ injury, such as pulmonary hypertension, in patients with sickle cell disease (SCD). Preservation of organ function is a critical criterion of the success of therapy. Nonmyeloablative allogeneic stem cell transplantation (NMA-SCT) is relatively non-toxic and frequently results in mixed hematopoietic chimerism. However, it is not known if mixed chimerism reduces end-organ toxicity. To assess the effects of mixed chimerism on reduction in hemolysis and endothelial function, sequential multiple parameters of intravascular hemolysis were measured in 3 SCD patients who developed mixed chimerism following NMA-SCT. All 3 reported significant improvement in SCD-related symptoms after transplant, despite mixed chimerism. We analyzed serum biomarkers, recently identified to be associated with pulmonary hypertension and endothelial dysfunction, and compared them to chimerism as measured by both routine genomic DNA analysis and by a erythroid lineage specific assay. These include markers of increased hemolytic rate (plasma hemoglobin (Hb), LDH, reticulocytosis), endothelial function (sVCAM-1), and decreased nitric oxide (NO) bioavailability (plasma NO consumption). Analysis of peripheral blood genomic DNA demonstrated 25–50% total donor cell engraftment from d41 to d180 in these patients. During this period of partial donor engraftment, all 3 patients demonstrated improvements in extent of intravascular hemolysis: Median pre to post NMA-SCT levels of LDH decreased 300 U/liter L (range 255–621 U/liter) to 162 U/L (range 122–199 U/liter), and of cell-free Hb concentration decreased from 11.8 uM (range 5.5–15.54) to 3.97 uM (range 1.61–7.75). Similarly, sVCAM-1 levels and NO consumption both decreased from median pre- to post NMA-SCT levels of 712.3 ng/ml (range 654.9–1067) to 413.9 ng/ml (193.9–608.2), and of 3.34 uM (1.91–7.63) to 1.32 uM (0.62–3.92), respectively. After d180, Pt 1 experienced graft rejection, and all hemolysis parameters again approached pre-transplant levels, demonstrating that reduction in intravascular hemolysis was directly attributable to the presence of circulating donor erythrocytes. Studies of RBC chimerism more accurately assessed the functional impact of donor hematopoiesis. RBC chimerism was serially measured by quantifying donor-derived beta-globin RNA in PBMC. Surprisingly, this revealed a ~ 2-fold higher level of donor erythroid precursor expression (70–100%) compared to the level of total donor cell engraftment, attributable to the improved survival of engrafted cells. This near-to full replacement of peripheral erythrocytes with donor derived RBCs accounts for the improvements in hemolysis. Our data reveal that levels of donor engraftment as low as 25% can ameliorate the degree of hemolysis and can potentially protect against ongoing chronic end-organ damage. These improvements have not been previously achievable with any other treatment for SCD, including hydroxyurea. Analysis of additional transplanted SCD patients is ongoing.