Identification of an Expanded Immature Cell Population in Human Bone Marrow during Erythroid Responses to ESA Treatment

Background and aim: Treatment with erythropoietin stimulating agents (ESA) is first-line treatment for anemic low-risk MDS patients, but although clinical variables such as endogenous serum erythropoietin levels have been associated with response, the bone marrow (BM) cell populations targeted during ESA-induced erythroid improvement have not been identified. Initiating SF3B1 mutations in MDS-RS arise in the multipotent hematopoietic stem cell (HSC), and we previously showed that most SF3B1-mutated MDS-RS patients retain a small residual wildtype HSC population, something that we propose may have clinical relevance (Mortera-Blanco, et al, Blood 2017). In this study, we aimed to map clonal dynamics of mutant and wildtype hematopoietic stem and progenitor cells (HSPCs) during steady-state and ESA treatment.

Methodology: We used advanced flow cytometry to sort and characterize HSPC populations in BM samples of SF3B1-mutated MDS-RS patients with stable anemia, before and during ESA response, and during chronic transfusion dependency. In addition to conventional HSPC markers, we added CD105 to the antibody panel as it has been reported to distinguish committed erythroid progenitors (Mori, et al, PNAS 2015). Variant allele frequency (VAF) of the known SF3B1 mutations was determined using droplet digital PCR.

Results: Expression patterns of mature CD38+ progenitors; common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs) and megakaryocytic-erythroid progenitors (MEPs), mostly followed published patterns with CD105 expression as expected in a subset of MEPs. The immature CD38- HSCs, multipotent progenitors (MPPs), and lymphoid-primed MPPs (LMPPs) should be negative for the CD105 surface marker (Mori, et al, PNAS 2015) but we confirmed a very low expression (<0.5% of CD34+CD38- compartment; median 0.15%; range 0.04 - 0.5%; n = 5) in normal BM samples and a similar frequency in MDS-RS with stable anemia (median 0.17%; range <0.01 - 1.12%; n = 9). However, in MDS-RS BM samples taken during ESA response, the CD34+CD38-CD105+ population increased up to 13 fold (range 2 - 13; n = 4). Interestingly, this population returned to the low baseline level, similar to normal BM, when the patients became refractory to ESA. While SF3B1 mutation burden in total BM mononuclear cells was not correlated with degree of anemia or treatment need, VAFs in CD34+ cells were significantly lower in patients with stable anemia compared to those with treatment need (p < 0.01). A similar pattern was observed in the mature CD34+CD38+ populations CMP, GMP and MEP (p < 0.05). Interestingly, in patients responding to ESA treatment, the erythroid-committed MEPCD105+ population was generally completely mutated, while the mutation burden in the expanding aberrant CD34+CD38-CD105+ population was much lower in several patients. Intriguingly, aberrant expression of CD105 was also found in mature erythroblast populations from ESA-responding MDS patients.

Conclusion: Our data indicate that presence of wildtype CD34+ BM populations in anemic MDS-RS patients is associated with better erythroid output and milder anemia. Our results show poor discriminative power of SF3B1 mutational burden in total BM as routinely assessed in the clinic, whereas SF3B1 VAF in defined CD34+ HSPC populations shows a strong correlation with treatment need. Expression of the surface marker CD105 identifies an immature CD34+CD38- BM population that expands during erythroid responses to ESA treatment. Because this population also can be detected at low levels in normal BM donors, and has a lower mutation burden than other HSPC populations in MDS-RS patients, we propose that this wildtype population acts as a reserve source of erythroblasts during stress erythropoiesis, possibly bypassing the traditional hierarchy.