Prospective subtyping identifies an association between high pretreatment levels of DEspR+ neutrophil extracellular trap-forming neutrophils and microvesicles during sterile inflammation with poor survival in patients with metastatic non-small cell lung cancer receiving first-line combination chemo-immunotherapy.

INTRODUCTION: High neutrophil-lymphocyte ratio (NLR) and neutrophil extracellular traps (NETs) are associated with poor overall survival in many cancers but are currently remain without safe effective therapy until subtype elucidation identifies a targetable molecular subtype whose inhibition will not compromise homeostatic pathogen defense. We tested the hypothesis that increased circulating NET-forming neutrophils (NET+Ns) expressing the cell-surface dual endothelin-1/signal receptor (DEspR) comprise a subtype that predicts poor survival outcomes in patients with metastatic non-small cell lung cancer (mNSCLC) receiving first-line chemotherapy-immunotherapy (chemo-IO), because peripheral levels of DEspR+ neutrophils and NET+Ns are increased in neutrophil-mediated secondary tissue injury during sterile inflammation across different diseases in which high NLR is associated with disease progression or exacerbation. METHODS: In a prospective observational study, we subtyped neutrophils by flow cytometry and NET+Ns by immunofluorescence cytology (IFC) confocal microscopy using fresh whole blood samples from 10 patients with mNSCLC at baseline and 6 weeks after the first dose of chemo-IO. We performed Kaplan-Meier survival analysis and Cox regression with Firth's penalty for 2-year overall survival (OS) and restricted mean survival time (RMST) analyses for 1-year OS. RESULTS: In 10 patients with mNSCLC, immunosuppressive PDL1+DEspR+ neutrophils and monocytes were increased at pretreatment baseline compared with normal levels. IFC detected increased circulating DEspR+citH3+[NET+Ns] and microvesicles (MVs), the majority of which were also PDL1+, and persisted 6 weeks after the first dose of chemo-IO. IFC of buffy coat smears detected DEspR+[NET+Ns] complexed with DEspR+EpCAM+ circulating tumor cells (CTCs). Concordant findings from Kaplan-Meier survival analysis and univariate Cox regression with Firth's penalty for 2-year overall survival (OS), and RMST for 1-year OS identified significant association between high baseline levels of DEspR+[NET+N] subtpe and overall survival respectively. CONCLUSIONS: These data identify the DEspR+ subtype of citH3+/PDL1+[NET+Ns] and MVs whose respective levels correlate with decreased 1-year and 2-year OS in patients with mNSCLC receiving first-line chemo-IO. Visualization of NET+N intercellular complexes in circulation depicts ongoing systemic inflammatory-immunosuppressive microenvironment in mNSCLC. As DEspR is cell-surface accessible and targetable, these data collectively provide a basis for further confirmatory research to evaluate DEspR as a potential actionable biomarker in larger and independent mNSCLC cohorts.