S162: LOSS OF HEMATOPOIETIC PROGENITORS HETEROGENEITY IS AN ADVERSE PROGNOSTIC FACTOR IN MYELODYSPLASTIC SYNDROMES

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Background: Myelodysplastic syndromes (MDS) represent a heterogeneous group of bone marrow disorders characterized by clonal evolution starting from the HSPCs compartment that contains the cell of origin for MDS. Stratification of patients at diagnosis to evaluate the risk of leukemic transformation is based on cytogenetic and molecular data. However, multiparametric flow cytometry (MFC) can also provide key information regarding not only MDS diagnosis but also for predicting the risk of leukemic evolution and guiding the choice of the therapeutic strategy. In this way, deciphering the diversity of the HSPCs compartment may allow the early detection of an emergent subclone that drive disease progression and could represent therefore an attractive tool to evaluate at diagnosis how close or far is the leukemic transformation. Aims: To explore the hematopoietic branching system, we designed a new MFC strategy which could be fully integrated in the MDS diagnostic workflow for predicting the outcome of these diseases. Methods: A total of 910 BM samples were collected between 2017 and 2022 in two different institutions (Cochin Hospital - University Paris Cite, n= 211; and Toulouse University Hospital, n=690). First, a retrospective cohort of 36 cryopreserved BM samples (27 MDS and 9 controls) was used to decipher the heterogeneity of the CD34+ HSPCs compartment using a panel of 19 markers and unsupervised analysis (t-SNE & Flowsom algorithms). A simplified MFC strategy (using 10 or 7 markers) was then applied to two independent prospective cohorts of fresh BM samples with or without MDS (cohort 1: 131 MDS and 44 controls; cohort 2: 510 MDS and 180 controls) and the Shannon entropy was calculated to measure HSPCs heterogeneity. For each cohort, normal value of HSPCs entropy was calculated on control samples. Normal or low level of HSPCs entropy in MDS samples was determined through z-score calculation (>-2 or ≤-2 respectively) compared to control samples. Thereafter, MDS features and progression-free survival (PFS) of patients with normal or low level of HSPCs entropy were analyzed. Results: Compared to controls, our unsupervised analysis of CD34+ HSPCs repartition revealed that a loss of HSPCs heterogeneity could be detected in most of MDS samples including low-risk MDS confirming therefore that quantification of HSPCs repartition could be highly relevant in these diseases. We then applied a similar strategy of HSPCs identification with a reduced number of markers to 2 independent larger cohorts of BM samples from patients with or without MDS. We observed a loss of HSPCs heterogeneity attested by a decrease of HSPCs entropy in respectively 47% and 39% of MDS cases. In both cohorts, a low HSPCs entropy was associated with a more advanced state of the disease with deeper cytopenias, higher R-IPSS risk and more somatic mutations. Interestingly, we found that the risk of MDS progression was significantly increased in patients with low level of HSPCs (3-years PFS: 44.4% vs 73.7%; p<0.0001). Above all, decreased HSPCs entropy still had a strong impact on PFS of patients with low or intermediate risk MDS (3-years PFS: 59.6% vs 96.6%, p<0.0001 and 47.7% vs 69.2%; p=0.0028 respectively). Finally, multivariate analysis confirmed that decreased HSPCs entropy was inversely correlated to the outcome of MDS independently of IPSS-R. Summary/Conclusion: Analysis of cellular HSPCs architecture in MDS represents a very powerful tool to identify MDS patients with a high risk of progression including from early stage of the disease, which could be useful for guiding the choice of specific therapeutic strategies.Keywords: Prognostic factor, MDS, Flow cytometry, Hematopoietic stem and progenitor cells

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