Single Cell Network Profiling (SCNP) Reveals Age-and Disease-Based Heterogeneity in Healthy Individuals and in Patients with Low Risk (LR) Myelodysplastic Syndrome (MDS)

Normal hematopoiesis changes with age through unknown mechanisms. Low risk (LR) myelodysplasia (MDS) is characterized by cytopenias arising through inefficient hematopoiesis. We hypothesized that both of these differences might result from changes in responsiveness to external signaling. To test this hypothesis, we utilized SCNP, a multiparametric flow cytometry-based assay that simultaneously measures both extracellular surface marker levels and changes in intracellular signaling proteins in response to extracellular modulators, quantitatively at the single cell level (Kornblau et al. Clin Cancer Res 2010).

SCNP was applied to examine baseline and intracellular signaling responses induced by the extracellular modulators EPO and GCSF in bone marrow (BM) mononuclear cells (BMMC) derived from healthy donors (n=15) and MDS (n=9) patients. The effects of donor age on signaling profiles in healthy BMMC were compared between samples collected by BM aspirate from 6 subjects aged 23–43 years (“younger”) and from the BM present in hip replacement samples from 9 subjects aged 54–82 years (“older”). Signaling profiles were also determined for 9 LR MDS patients aged 53–83 years and compared to the age-matched healthy “older” control. Metrics used for analysis included fold change, total phosphorylation levels, and the Mann-Whitney U statistic model.

There were no differences in the frequency of CD34+ cells (R²= 0.006, p= 0.78) between “younger” and “older” healthy donor samples, suggesting any differences observed in signaling would likely be due to donor age rather than sample source. There was no age-related difference in functional signaling ability in response to GCSF-induced p-STAT1, p-STAT3, & p-STAT5 levels; however, early erythroblasts and normoblasts from healthy “older” donors were significantly less responsive to EPO, as measured by induced phospho (p)-STAT5 levels than those derived from healthy “younger” donors (e.g. R²= 0.654 p=0.008 for erythroblasts and R²=0.628 p=0.0004 for normoblasts). Signaling profiles classified Refractory Anemia with Excess Blasts (RAEB) patients into 2 categories based on differences in EPO- and GCSF-induced signaling (Table 1). Compared to age-matched healthy “older” controls, one subset was characterized by a high % of RBC precursors (CD45lo nRBC) and increased p-STAT5 levels in response to EPO and the other subset by a high % of myeloid cells with robust GCSF-induced p-STAT3 & p-STAT5 responses in both total myeloid and CD34+ cells. By contrast, patient samples with Refractory Anemia with Ringed Sideroblasts (RARS) had a high % of CD45lo nRBC but lacked robust p-STAT5-induced signaling after modulation with EPO.

Overall, these data show the feasibility of using the SCNP assay in BM samples to functionally characterize signaling pathways simultaneously in different cell subsets of healthy donors and patients with MDS. In healthy individuals, age-related differences in EPO signaling were discovered. In LR MDS, differences in signaling were observed between cases and in comparison to the data from healthy controls. Deciphering signaling profiles in healthy donor versus MDS patient samples may result in improved, biologically-based disease classification that informs more effective patient management. The clinical relevance of these findings in terms of disease course and treatment is currently under investigation.

Cleary Cohen:Nodality Inc.: Consultancy, Equity Ownership. Huang:Nodality Inc.: Consultancy, Equity Ownership. Cesano:Nodality: Employment, Equity Ownership. Hawtin:Nodality: Employment, Equity Ownership. Ware:Nodality Inc.: Employment, Equity Ownership.