478P Explainable machine learning analysis reveals neutrophil dynamics as predictor of immunotherapy response in advanced NSCLC

Background:The SPACETIME consortium, an EU-funded research project under the Horizon Europe Program (Mission Cancer) and formed by 15 partners, investigates the spatial and temporal evolution of the tumor microenvironment in NSCLC.Here we report the preliminary clinical-molecular characterization, functional assays, and transcriptomic profiling of the non-squamous NSCLC samples selected so far for the SPACETIME project.Methods: Baseline samples from patients with KRAS-mut or pan-wt non-squamous NSCLC (fresh and FFPE tissue, plasma, and malignant effusions) were collected in five hospitals of the Barcelona area from August 2024 to December 2025.Molecular characterization of FFPE samples included a 30-gene DNA next-generation sequencing (NGS) panel and PD-L1 IHC.Plasma samples were analyzed using a 19gene DNA NGS panel.Both FFPE and plasma samples were also analyzed by nCounter using RNA Immune (n=579 genes) and Circular (n=78 genes) panels.Primary cultures for drug testing were established from malignant effusions and fresh tissue.Results: Forty-nine patients were screened, and twenty were selected for the SPACETIME project (Stages IA-IVB).Twelve patients were KRAS-mut and 8 pan-WT.ROS1 mRNA overexpression was observed in 73% of early-stage (n=8/11) and 22% of advanced (n=2/9) tissue samples; being particularly frequent in KRAS-mut tumors (n=9/12).Fresh tissue or pleural effusions were obtained in 8 cases (41%), and two primary cultures successfully established.One of them was treated in vitro with gemcitabine-carboplatin and synergistic activity was observed, consistent with clinical benefit.RNA profiling of tissue (n=16) and plasma (n=18) samples revealed distinct messenger and circular RNA patterns.The fresh or FFPE samples collected will also be submitted to spatial transcriptomics, proteomics, glycomics and metabolomics by different members of the SPACETIME consortium. Conclusions:This study shows the feasibility of comprehensively characterizing nonsquamous NSCLC using next generation sequencing, functional, and transcriptomic techniques.ROS1 expression differences across stages indicate that it may contribute to disease progression.