PF585 GENE-EXPRESSION PROFILE OF HIGH-DENSITY NEUTROPHILS REVEALS PROGRESSIVE DIFFERENCES IN MGUS AND MULTIPLE MYELOMA, ASSOCIATED WITH REDUCED PHAGOCYTOSIS AND INCREASED INFECTION SUSCEPTIBILITY
Authors: A. Romano, N.L. Parrinello, V. Simeon, C. Bellofiore, C. Giallongo, F. Puglisi, P. La Cava, G. Camiolo, M.V. Brundo, S. Silvia, D. Tibullo, G.A. Palumbo, C. Conticello, P. Musto, F. Di Raimondo
Journal: HemaSphere (2019)

Background: In Multiple Myeloma (MM) the deficiency of the immune system facilitates tumor progression as well as failure to drug therapy and results in high rate of infections. Also asymptomatic patients affected by monoclonal gammopaty of unkwown significance (MGUS) or smoldering myeloma (SMM) have a 2-fold risk of developing a bacterial and viral infections compared to matched controls. Aims: To understand neutrophil impairment in the progression from MGUS through active MM, we investigated the function of mature, high-density neutrophils (HDN) at the steady state. Methods: HDN were isolated from peripheral blood by a combination of immunomagnetic and density gradient centrifugation. In 7 MM, 3 MGUS and 3 healthy subjects we performed gene expression profile (GEP) using Illumina HumanHT-12 v4 bead arrays. Findings were then validated by RT-PCR, flow-cytometry and immuno-fluorescence in a series of 60 newly diagnosed MM (43 treated according the VTD schedule) and 30 MGUS patients evaluated between January 2013 and December 2014. Results: The direct pair-wise comparison identified a total of 894 differentially expressed genes: 551 upregulated and 343 downregulated (FDR<5%) in MM-HDN compared to healthy HDN. We identified a total of 3182 differentially expressed genes between MM and MGUS HDN: 1491 upregulated and 1691 downregulated (with 133 genes 3-fold upregulated). Gene set enrichment analysis (GSEA) identified dysregulation in chemokine signaling pathway and FC-gamma receptor mediated phagocytosis, leukocyte trans-endothelial migration, regulation of actin cytoskeleton, adherens junction, chemokine signaling pathway and FC-gamma receptor mediated phagocytosis. By flow-cytometry we found that the inducible FcγRI (also known as CD64) was higher in MM than in MGUS and healthy HDN, resulting in a reduced phagocytic activity and oxidative burst. MM-HDN and MGUS-HDN clustered separately: only 390 genes were progressively increased in the progression from MGUS through MM, suggesting a myeloid dysfunction supporting MM progression. High-expression genes in MM-HDN were enriched for interferon alpha response, interferon gamma response, IL-6-JAK-STAT3 pathway, and inflammatory responses. Presence of higher both STAT-3 and STAT-6 regulated transcripts was validated increased phosphorylation of STAT-3 and STAT-6 in freshly collected sorted primary samples. One of the most affected genes progressively up-regulated from healthy through MGUS and MM-HDN was ARG1, a transcriptional target of both STAT-3 and STAT-6 ARG1, that our work previously showed increased in MM-PMN-MDSC, associated to bortezomib refractoriness (Romano, 2018; Giallongo, 2016). Indeed, ARG1+HDN were immune-suppressive in allogenic co-culture in vitro. In 43 consecutive newly-diagnosed MM patients, candidates to autologous stem cell transplantation, who received first-line treatment based on bortezomib, thalidomide and dexamethasone (VTD), high CD64 could identify at diagnosis patients with inferior median overall survival (39.5 versus 86.7 months, p = 0.04), while CD16 could not. Summary/Conclusion: In clinical progression through MM, neutrophils may play a role in supporting both the increased susceptibility to infection and the immunological dysfunction that leads to tumor progression.