Eltrombopag Specifically Expands Hematopoietic Multipotent Progenitors in Human Aplastic Anemia

Eltrombopag (EPAG), a thrombopoietin receptor agonist, has been recently added to first-line immunosuppressive therapy (IST) in immune aplastic anemia (AA), significantly improving hematologic recovery and survival (Townsley et al. NEJM 2017), corroborating preclinical data suggesting a direct effect of EPAG on hematopoietic precursors. Here, we analysed the in vivo effects of EPAG on the composition of the hematopoietic stem and progenitor cell (HSPC) compartment after hematologic recovery in human AA patients treated with IST combined with EPAG.

Twelve patients with severe immune aplastic anemia who achieved hematologic response six months after initial treatment were invited to participate in the study. Six patients were treated with EPAG associated with cyclosporine and six were treated with rabbit antithymocyte globulin and cyclosporine, but treatment choice was not determined by this study. 25 mL of marrow aspirate was obtained from each patient for multiparametric analysis in an 8-color flow cytometry. Mononuclear cells were separated by gradient density centrifugation and CD34+ cells were purified using magnetic column beads. We used a 9-antibody panel to identify HSPC subpopulations, as follows: hematopoietic stem cells (HSC), CD34+ CD38- CD45RA- CD90+ CD49f+; multipotent progenitor cells (MPP), CD34+ CD38- CD45RA- CD90- CD49f-; multilymphoid progenitor cells (MLP), CD34+ CD38+ CD45RA+ CD10+ CD7-; common myeloid progenitor cells (CMP), CD34+ CD38+ CD45RA- CD135+ CD10- CD7-; granulocyte-monocyte progenitor cells (GMP), CD34+ CD38+ CD45RA+ CD135+ CD10- CD7-; and megakaryocytic-erythroid progenitor cells (MEP), CD34+ CD38+ CD45RA- CD135- CD10- CD7-. Purified marrow CD34+ cells were also assessed in methylcellulose medium and colonies were counted 14 days after triplicate plating. Cell populations and colony formation assay were compared between the two groups using a two-tailed non-parametric Mann-Whitney test.

The percentage of CD34+ of total nucleated bone marrow cells was 1.2% (range, 0.28-2.9%) in patients who received IST/EPAG and 0.2% (0.11-0.65%) in those who received IST alone (P<0.005). The percentage of MPPs cells was the only one significantly expanded in patients who received IST/EPAG as compared to IST alone (P<0,05). There was no statistical difference in the number of colony-formation units between groups.

Townsley et al. (NEJM 2017) reported on the CD34+ compartment reconstitution three and six months after initial therapy, but did not compare this recovery to patients not receiving EPAG. Additionally, they did not indicate which primitive cells are directly affected by EPAG. It was speculated that EPAG may act on HSPC proliferation via the THPO-MPL signalling pathway, supported by previously evidence that loss-of-function mutations in the THPO gene result in aplastic anemia (Dasouki et al. Blood 2013).

Here, we compared for the first time the HSPC compartment reconstitution in AA patients treated with either IST alone or EPAG added to IST. We found that marrow CD34+ cell numbers were higher when EPAG was added to IST, indicating a positive effect on the HSPC compartment. More importantly, we found that MPPs were the only HSPC subset to be significantly increased when patients were treated with EPAG. The role of the THPO-MPL signalling pathway in HSPCs is not entirely clear, but our findings are in agreement with recent observations. In xenotransplant models, Matsuoka et al (Cell Transplantation 2017) found that human CD34+ MPL- cells are able to sustain long-term engraftment, whereas MPL+ cells showed deficient repopulation capacity. Likewise, Cocault et al (Experimental Hematology 2016) found that in human CD34+CD38- cells, MPL is preferentially expressed in CD90-CD49f- cells. Recent observations in refractory AA patients suggest that EPAG may expand quiescent preexisting abnormal clones in the marrow (Winkler et al. Blood 2019). However, in our cohort, we did not observe any aberrant HSPC immunophenotype suggesting the expansion of abnormal clones.

In conclusion, we found that in patients with immune aplastic anemia, EPAG significantly expands CD34+ cells and specifically expands hematopoietic multipotent progenitor cells. Our findings support the hypothesis that in vivo the THPO-MPL signalling pathway plays a role in human MPP proliferation, but not in HSCs.