Cytokine profiling identifies an IL‑8/IL1RAP‑Pathway–Linked inflammatory phenotype in myeloproliferative neoplasms (MPN) associated with high symptom burden and poor outcomes

Abstract

Abstract Background Chronic inflammation, characterized by elevated proinflammatory cytokines within the bone marrow (BM) microenvironment and peripheral blood (PB), is a hallmark of MPNs and may drive progression to secondary AML (sAML; Tefferi A, Blood 2011; Fleischman AG, Blood 2011). IL1 receptor accessory protein (IL1RAP) amplifies IL1 cytokine signaling, is selectively overexpressed on MPN stem/progenitor cells, and contributes to disease propagation and therapy resistance. The relationship between IL1RAP-related pathways, inflammatory networks, symptom burden, and outcomes across MPN subtypes remains unclear. We profiled plasma cytokines to identify IL1RAP-driven inflammatory phenotypes and their clinical associations. Methods Plasma from 39 individuals (9 healthy donors [HD], 17 MPN, 13 sAML) collected between July 2022 and March 2025 was analyzed for over 20 cytokines using a Luminex 30-Plex assay. An IL1RAP composite score was derived by standardizing concentrations of IL1RAP-mediated cytokines (IL1RA, IL6, IL8, MCP1, IL10) into z-scores and averaging values. Hierarchical clustering and K-means (k=3) defined inflammatory phenotypes, with principal component analysis confirming cluster separation. Clinical variables included DIPSS, MIPSS70+, driver and high-risk mutations, JAK inhibitor (JAKi) exposure, transplant status, and the MPN Symptom Assessment Form (MPN-SAF) Total Symptom Score (TSS). Associations between cytokines, clusters, and symptoms were tested using Spearman and Kruskal-Wallis. Results Median ages were 68 years (MPN), 67 years (sAML), and 32 years (HD). Driver mutations included JAK2 (62%), CALR (23%), MPL (10%), and triple-negative (5%). TP53 and ASXL1 mutations were present in 10% and 12%, respectively, and were more frequent in the high-inflammation cluster, although not statistically significant (p=0.54). DIPSS categories were low (n=4), intermediate-1 (n=6), intermediate-2 (n=5), and high (n=2); 35% were high risk by MIPSS70+. At sampling, 13% were on JAKi, 46% had prior JAKi exposure, and 21% (4/19) underwent alloHCT. IL8 was significantly elevated across groups (p=0.0014, FDR=0.0098; range ~40–>200 pg/mL), highest in sAML and MPN with the IL1RAP inflammatory phenotype (Cluster 3). IL6, IL1RA, and MCP1 trended higher in patients with adverse features, including high DIPSS/MIPSS70+, TP53/ASXL1 mutations, and sAML. IL1RAP composite scores (range 0–+1.1) were highest in patients with adverse features and correlated with high-inflammation clusters. MPN-SAF TSS correlated with IL8 (ρ=0.73; p=0.0009) and IL1RA (ρ=0.60; p=0.011). Cytokine profiles, including IL8, IL1RA, IL6, MCP1, and IL10, defined three phenotypes. Cluster 1 (low inflammation, n=12) was characterized by IL8 levels of approximately 40 pg/mL, an IL1RAP score near 0, low or intermediate DIPSS, and the lowest MPN-SAF scores (median 16.5); all transplanted patients (4/12) were in this group. Cluster 2 (intermediate inflammation, n=1) had IL8 levels near 120 pg/mL, an IL1RAP score of +0.5, an MPN-SAF score of 19, and no patients underwent transplant. Cluster 3 (high inflammation, n=4) exhibited IL8 levels between 120–200 pg/mL, an IL1RAP score of +1.1, high DIPSS/MIPSS70+ categories, and the highest MPN-SAF scores (median 34); no patients in this cluster underwent transplant. AML progression was more frequent in Clusters 2 and 3 (60% in Cluster 3) compared with 30% in Cluster 1 (p=0.03). Conclusion Cytokine profiling integrating clinical and molecular features, an IL1RAP composite score, and symptom assessment identified an inflammatory phenotype driven by IL8, IL1RAP-related cytokines, and other pathways. This phenotype, predominant in Cluster 3, was enriched in sAML and high-risk MPN, associated with high symptom burden and failure to proceed to alloHCT, a scenario reflecting aggressive disease or ineligibility for curative therapy. Heightened inflammation was linked to worse disease burden and AML transformation, potentially hindering alloHCT. The IL1RAP composite score defines a distinct cytokine phenotype that may be predictive or prognostic of adverse outcomes and warrants prospective validation.