From chaos to clarity? decoding the inflammatory profiles in lower-risk MDS Free

Immune dysregulation is increasingly recognized as both a pathogenic driver and therapeutic target in Myelodysplastic Syndromes/Neoplasms (MDS). Despite improvements in prognostic stratification by the Molecular International Prognostic Scoring System (IPSS-M), MDS patients remain heterogeneous in their clinical behaviour, treatment responses and disease evolution. Inflammation impact seems to be preponderant in pathophysiology of lower risk (LR)-MDS. By assessing 36 analytes in LR-MDS serum at diagnosis with a screening panel, we stratified and characterized LR-MDS cases into biologically distinct subgroups with different clinical characteristics and treatment response to erythropoietin stimulating agents (ESAs).

We evaluated 95 MDS cases selected per lower risk IPSS-R (score < 3.5), and symptomatic anemia, analyzing 36 serum proteins (cytokines, alarmins, soluble receptors and growth factors). Complete clinical annotations (including IPSS-M scores) were collected. Serum protein levels were assessed using custom Luminex Screening Assay panels. For bioinformatic analysis, proteins were regrouped into simplified biologically relevant groups: pro-inflammatory (S100A8/A9, TNF-α, IL-1β, IL-18, IL-17E, IL-8, IFN-α/γ, CCL2/3/4, RANTES, CCL11, CXCL9/10/7/4; n=18), anti-inflammatory (IL-1RA, IL-10; n=2), bimodal (pro- and anti-inflammatory potential; IL-6, IL-4, IL-13; n=3), growth factors (VEGF, PDGF-BB, HGF, FGF-BASIC, M-CSF; n=5), receptors (TNFR1, CD25, GP130; n=3), apoptosis-related (TRAIL; n=1), and immune expansion (IL-2, IL-5, IL-7, IL-15; n=4). Following log-transformation and z-score normalization, we performed principal component analysis (PCA) and k-means clustering to identify distinct LR-MDS subgroups. Feature selection by random forest models identified the most discriminatory cytokines in LR-MDS. Subgroups were compared in terms of inflammatory state, clinical variables, and clinical endpoints including overall survival (OS) and ESA response duration.

Median age at diagnosis was 74 years (range 47-91) (M:F ratio 1.6:1). Per WHO 2022 classification (n=69): MDS-LB = 44, MDS-SF3B1 = 21, and MDS-del(5q) = 4. IPSS-R scores (n=95) were: very low = 31, low = 55, intermediate = 9. IPSS-M scores (n=62): very low = 16, low = 32, moderate-low = 9, moderate-high = 4, and very high = 1. Through PCA we identified five distinct clusters based on serum protein levels. Cluster sizes were: C1 (n=18), C2 (n=17), C3 (n=12), C4 (n=22), and C5 (n=26). Inter-cluster comparisons revealed significant differences between all analytes annotated for biological function (adj. p < 0.001, except for receptors and apoptosis-related groups), especially for bimodal and pro-inflammatory proteins, and growth factors. Random forest analysis identified IL-4, IL-17E, IL-2, CCL3, IL-1β, IL-7, CCL4, IL-13, IFNγ and CXCL7 as most discriminatory serum proteins.

Cluster-specific characteristics: C1 (72% low IPSS-R, median age 76 years) showed low pro-inflammatory levels (mean log10: 1.88); C2 had the highest MDS-del(5q) frequency (18%) and highest inflammatory burden (mean log10: 2.30, 3.2-fold higher than C3); C3 was the youngest (median age 58.6 vs 76.6 years overall, p=0.041), enriched for MDS with ring sideroblasts (MDS-RS, 33%), and displayed the lowest pro-inflammatory profile (mean log10: 1.80).

There was a numerical difference in rate and duration of ESA response, with a trend to longer ESA response in C4 and C5 (log-rank p=0.089). Interestingly, clusters C1, C2 and C3 had higher rate of resistance or early relapse to ESA therapy (56%, 50%, and 67%, respectively) when compared to clusters C4 and C5 (18% and 23%, respectively). While overall survival did not significantly differ across clusters, cluster C2 showed higher rate of AML transformation (14% vs 6% non-C2).

By profiling serum analytes in 95 LR-MDS patients we identified five biologically distinct LR-MDS subgroups with differential inflammatory signatures correlating with ESA responses. Extreme pro-inflammatory profile (C2), and low inflammatory profiles (C1 and C3) identify LR-MDS cases refractory to ESAs. In C2 poor response may be correlated with a more severe disease course, with AML early progression; while C3 is enriched for young MDS cases with low-inflammation burden with MDS-RS. These results are relevant to support early therapeutic decision-making and prompt further studies in larger LR-MDS cohorts to dissect cytokine-mediated molecular pathways.