Searching for a myelodysplastic syndrome stem cells (MDS-SCs) signature by flow cytometry: Toward a new prognostic tool?

Abstract Introduction Myelodysplastic Syndromes (MDS) are a group of clonal myeloid disorders characterized by poor prognosis and frequent progression to Acute Myeloid Leukemia (AML). Several studies focused on the properties of malignant stem cells that drive AML pathogenesis; on the contrary, relatively little attention has been paid to MDS. It is hypothesized that the disease arises from a pool of immature malignant stem and progenitor cells (MDS-SCs), serving as a reservoir for disease evolution and progression. Studies demonstrated that these cells showed a similar phenotype to Hematopoietic Stem Cells (HSCs) but also expressed markers associated with AML such as CD123, CD371 (CLL1), and CD366 (TIM-3). Based on these premises, our study explored the whole pool of CD34+ cells, focusing on both compartment CD34+CD38-CD90+ HSCs and CD34+CD38-CD45RA+ LSCs, with a twofold aim: 1)to investigate if LSCs AML-like cells are already present in MDS samples and 2)to identify a specific MDS-SCs phenotype (HSCs-like) in our cohort of patients (pts). Our ultimate intent is to develop a new tool that could help to improve prognosis and management of MDS pts. Methods: Bone marrow (BM) samples from pts with a suspected diagnosis of MDS have been blindly collected and a flow-cytometry antigen panel, including CD34, CD38, CD90, CD45RA, CD123, CLL-1 and TIM-3, has been created. Flow-cytometry analysis has been performed considering the whole CD34+ cells compartment, and distinguishing the CD34+CD38- subset (comprising HSCs, MPP and LSCs) from the CD34+CD38+ subset (comprising MEP, CMP and GMP). Meanwhile, cytomorphology, histopathology, cytogenetic and molecular tests have been conducted for diagnostic confirmation and risk stratification based on the Revised International Prognostic Scoring System (IPSS-R).ResultsBone marrow samples from 55 pts, median age 74ys, sex ratio male/female 37/18, have been collected; in 48/55 (87%) pts diagnosis of MDS was confirmed by morphology (cytomorphology of BM aspirate and biopsy), percentage of blasts and /or cytogenetics alterations, while 7/55 (13%) were considered “undefined MDS” due to the lack of a definitive diagnosis. By using a strict flow-cytometry gating strategy we investigated the CD34+CD38- cells compartment, focusing on two different subsets CD34+CD38-CD90+ HSCs and CD34+CD38-CD45RA+ LSCs. We have been able to stratify the 55 MDS patients in 4 groups, based on presence or absence of these two types of cells. 17/55 (31%) MDS [group 1] were negative for both HSCs and LSCs, and this group comprise: 9 IPSS-R low, 2 IPSS-R int, 2 undefined MDS. 5/55 (9%) MDS[group 2] were positive just for HSCs, with 2 IPSS-R low, 1 IPSS-R int and 1 undefined MDS. 10/55 (18%) patients [group 3]were double positive, presenting both HSCs and LSCs; they comprise 1 IPSS-R low, 2 IPSS-R int, 2 IPSS-R high and 1 undefined MDS. Finally, 23/55 (42%) patients[group 4]presented with just LSCs: this group comprises 5 IPSS-R high, 3 IPSS-R int, 12 IPSS-R low and 3 undefined MDS. Moreover, in this group 6/23 (26%) MDS patients evolved to AML. For 7/55 (13%) MDS patients IPSS-R scores are still under investigation. Up until now, in 17/55 (31%) an NGS sequencing was performed, resulting in known mutations of the RNA splicing mechanism (SRSF2, SF3B1, U2AF1), DNA methylation (IDH2, TET2, DNMT3A) and DNA transcription (KRAS, RUNX1), plus mutations in histone modification (EZH2) and chromatid cohesion (STAG2). Interestingly, in group 1 and 2 we have identified initiating MDS mutations, while in group 3 and 4 AML-like mutations were already present. Conclusions In order to clarify MDS pathogenesis, we developed a flow-cytometry strategy to stratify pts based on the stem cell compartment. We have been able to identify those pts already presenting LSCs AML-like cells that are more likely to evolve to AML [groups 3 and 4] and differentiate them from pts that instead still present an MDS-like phenotype, with NGS initiating mutations [groups 1 and 2]. By combining advanced flow-cytometry assays with NGS and cytogenetic analysis, we may help clinicians to enlighten the pathogenesis of MDS from the initial key events, through the identification of MDS-SCs that, unlikely to normal HSCs, start accumulating mutations. For this reason, we will keep enlarging our cohort and compare it to a dataset of normal BM. Moreover, this tool may help improving the monitoring of those pts that are more likely to evolve to AML in a short period of time.