PS1335 ANALYSIS OF REACTIVE OXYGEN SPECIES IN MDS HEMATOPOIETIC STEM AND PROGENITOR CELLS AND THEIR CORRELATIONS WITH CLINICAL DATA

Background: Myelodysplastic Syndromes (MDS) are an heterogenous group of clonal hematopoietic stem cell malignancies. Previous studies showed that reactive oxygen species (ROS) play a role in the pathogenesis and clinical evolution of MDS (Novotna B, Leuk Res, 2009; Chung, Y. J., Leuk Res, 2014), contributing to hematopoietic stem and progenitor cells (HSPC) genetic instability. Less is known about ROS levels in the different sub‐populations of MDS HSPC and how they correlate with clinical data in MDS patients. Aims: Our study aims to analyze ROS levels in MDS hematopoietic stem cells (HSC), common myeloid progenitors (CMP), granulocyte macrophages progenitors (GMP) and megacaryocyte‐erythrocyte progenitors (MEP); in addition, we want to investigate the relationship between ROS levels and clinical data. Methods: Thirty‐eight MDS (Table 1) and 27 normal bone marrow (NBM) were collected. In order to avoid extra stress to cells, samples were run for analysis 1 hour after bone marrow aspiration, with no further CD34+ cells enrichment procedure. ROS levels were assessed using fluorescent dye CellROX ® (TermoFischer) and quantified by fluocytometry as mean fluorescence intensity (MFI): for each sample, ROS values in lymphocytes were used to normalise ROS levels in the other subpopulations. Multiparameter flow cytometry was used to identify HSC, CMP, GMP and MEP, through CD34, CD38, CD45RA, CD123 and CD90 surface levels (Manz GM, PNAS, 2002). Results: Among Lineage negative (Lin‐) CD34+CD38+ cells, the percentage of CMP was 42%, 26.8% and 35.7% for MDS with no excess blasts (MDS no EB), MDS with excess blasts (MDS‐EB) and normal bone marrow (NBM) respectively (MDS no EB vs MDS EB p:0.003) . GMP were 17%, 29.5% and 18.7% for MDS‐no BE, MDS‐BE and NBM (MDS no EB vs MDS EB p:0.017 ). No differences were found in percentages of MEP between MDS and NBM. ROS levels were higher in GMP than in the other progenitors, regardless the sample group ( p  <  0.01 ). In IPSS low risk MDS, CMP and GMP had higher ROS levels compared to NBM CMP and GMP (2.36 and 3.17 vs 1.81 and 2.58, p: 0.025 and p:0.041 ). Hematopoietic stem cell (HSC) percentages on Lin‐ CD34+CD38‐ cells were more elevated in MDS than in NBM (2.5% vs 1.3%, p:0.038 ); in particular, MDS‐BE showed a higher HSC ratio compared to NBM (3.5% vs 1.3%, p:0.023 ). HSC ROS levels did not differ in MDS and NBM. Regarding clinical data, we found a direct correlation between ROS levels in CMP, GMP and MEP of low and intermediate‐1 (int‐1) risk MDS and absolute neutrophil count (ANC), the correlation was especially strong in GMP ( p:0.01 ). Similar results were found between ferritin values and ROS expression in CMP (p:0.024), MEP ( p:0.02 ) and GMP ( p:0.004 ). Concerning treatments, GMP from low/int‐1 risk patients that lost response to Erythropoietin tended to show higher ROS levels ( p:0.05 ). A similar correlation, although not yet significant, was seen in platelets count vs ROS levels on CMP, GMP and MEP. In our study, overall survival was not influenced significantly by ROS levels, but a tendency to show higher ROS in “long survivor” patients was spotted. Summary/Conclusion: Our data confirm that ROS play a role in low and int‐1 risk MDS. The positive correlations between ROS levels and clinical data as ANC and platelet count suggest that the MDS HSPC are subject to considerable stress as they struggle to maintain an effective hematopoiesis. Future studies should focus on how to reduce oxidative stress in HSPC: it might be a cause of increased genetic instability especially in GMP, already known to be the expanded population in AML (Goardon N, Cancer Cell, 2011).