Circulating low-density neutrophils as biomarkers of resistance to first-line anti-PD-1/PD-L1 immunotherapy in non-small cell lung cancer

BACKGROUND: Immune checkpoint inhibitors (ICIs) have improved outcomes in advanced non-small cell lung cancer (NSCLC), however reliable predictive biomarkers are lacking. Our group previously reported an association between high levels of circulating low-density neutrophils (LDNs) and resistance to ICI monotherapy. We present updated results, including a validation cohort, proteomic characterization of LDNs, and in vivo experiments exploring mechanisms of resistance. METHODS: NSCLC patients treated with first line ICI monotherapy (n=60) or combined with chemotherapy (CT+ICI) (n=60) were recruited. LDNs were quantified by flow cytometry and correlated with clinical outcomes. Phenotypes of LDNs and conventional neutrophils were characterised by flow cytometry and quantitative proteomics. Plasma cytokine measurements and in vivo experiments were conducted to assess the role of LDNs in ICI resistance. RESULTS: High baseline LDN levels were significantly associated with primary resistance to ICI monotherapy, with patients showing an overall response rate (ORR) of 17% vs 50% (p=0.04) and median progression free survival (mPFS) of 2.3 months vs 21.8 months (p < 0.001). No such association was seen in patients treated with CT+ICI, showing a LDN depletion in responders. LDNs exhibited an aged phenotype and distinct proteomic profile. Plasma from high-LDN patients showed elevated myeloid-expansion (M-CSF, IL1β) and inflammatory cytokines (CXCL9, IL-25). Depletion of Gr1+ population enhanced response to ICI and CT+ICI in the Lewis Lung Carcinoma (LLC) model with high LDNs. CONCLUSION: High baseline LDNs predict resistance to ICI monotherapy in NSCLC and combination with chemotherapy may overcome this resistance. Additional therapeutic strategies targeting LDNs could enhance immunotherapy efficacy.