Citrullinated histone H3 and elevated polyamine metabolism gene expression in peripheral blood polymorphonuclear leukocytes as biomarkers for poor immunotherapy response in cancer patients

There is a critical need to identify validated biomarkers that can predict cancer patient outcomes. This study evaluated biomarkers of neutrophil extracellular traps (NETs) and polyamine gene expression in peripheral blood that may correlate with greater risk for poor response to immunotherapy. Peripheral blood collected from cancer patients being treated with chemo/immunotherapy and from healthy volunteers was analyzed for levels of citrullinated histone H3 (Cit H3, a NETosis marker), inflammatory cytokines, and circulating tissue factor positive microparticles (TF + MPs). Elevated levels of plasma TF + MPs, Cit H3, IL-8, and IL-6 each significantly correlated with poor response to immunotherapy. Since in vitro stimulation of both human and mouse neutrophils to form NETs was blocked with addition of an inhibitor of polyamine uptake (Trimer44NMe), RNA expression of polyamine/glutamine metabolism genes was evaluated in both peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMN). Elevated expression of glutamine-associated genes in PBMCs and decreased expression of polyamine-associated genes in PMN correlated with favorable patient response to immunotherapy. Although not reaching significant levels, a high overall polyamine score in peripheral PMNs as well as increased plasma Cit H3 levels correlated well with poor response to immunotherapies in cancer patients. In a mouse model of triple negative breast cancer, polyamine blocking therapy inhibited not only primary tumor growth and lung metastases but also decreased intratumor NETs. Results from this study suggest a mechanistic link between NETosis and polyamine gene expression in PMN that warrant further investigation as peripheral blood biomarkers for immunotherapy response.