Abstract 7146: Unmasking host immune complexity: Single-cell transcriptomic analysis of circulating PBMCs in non-small cell lung cancer treated with immunotherapy based first line - I3LUNG study

Abstract Background and Purpose: Biomarkers for immunotherapy in NSCLC remain inadequate, with PD-L1 continuing to underperform despite its ongoing use. "Ghost biomarkers", such as circulating immune profiling, hold potential to reveal host immunity dynamics and identify ways to enhance immune fitness for improved therapeutic outcomes. This study uses single-cell technologies to analyse circulating immune cells in stage IV NSCLC patients (pts) receiving first line mono-immunotherapy (IO) or chemo (CT)-IO, aiming to identify subpopulation who might benefit or not from treatment. Methods: Blood samples from 33 stage IV NSCLC pts were collected at the baseline of first-line IO or CT-IO, as part of the Horizon Europe-funded I3LUNG study (NCT05537922) conducted at IRCCS Istituto Nazionale dei Tumori of Milan. Single-cell RNA sequencing was performed by Chromium Single Cell 5’ v2 kit (10X Genomics) on PBMCs, enriched for neutrophils sorted by FACS (CD66b+CD15+). After quality control, 30 samples were included in the analysis. Results: Among 30 pts, 27 were treated with CT-IO and 3 with mono-IO, 25 were adenocarcinoma, 4 squamous and 1 NOS carcinoma. UMAP analysis revealed distinct "swan neck" clusters of immature neutrophils, predominantly expressing LCN2 gene, in pts with disease progression (p-value <0.05, logFC -3, 67). These cells were notably absent in pts with complete response or partial response. Conversely, responders showed the enrichment of a mature subset of IGHM TCL1A, IGHD expressing B cells ("smart” B cells) (all genes with p-value <0.05 and logFC >2.5). Of note, TCL1A gene is involved in the formation of tertiary lymphoid structures, which are positively correlated with IO responses. Although based on a very small cohort in which no progressive disease occurred, we observed T and NK cells enrichment in mono-IO treated pts with high PD-L1 expression. Conclusions: To our knowledge, this is the largest prospective cohort that analyses circulating single-cell PBMCs in stage IV NSCLC pts treated with a first-line IO based. We identified novel circulating immune subsets, such as "swan neck" neutrophils and "smart” B cells, which emerged as promising host biomarkers. These subsets consistently acted as negative and positive predictors, respectively to first-line IO-based therapy. Citation Format: Arsela Prelaj, Monica Ganzinelli, Alessandro Guidi, Chiara Cavalli, Cecilia Silvestri, Antonino Belfiore, Laura Mazzeo, Claudia Proto, Mario Occhipinti, Marta Brambilla, Teresa Beninato, Leonardo Provenzano, Luca Inverizzi, Giulia Bertolini, Daniele Lorenzini, Filippo De Braud, Luca Agnelli, Andrea Vingiani, Sabina Sangaletti, Giuseppe Lo Russo. Unmasking host immune complexity: Single-cell transcriptomic analysis of circulating PBMCs in non-small cell lung cancer treated with immunotherapy based first line - I3LUNG study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7146.