Integrated genomic and immunophenotypic profiling reveals monoclonal origin, smoking-driven evolution and...

Pulmonary adenosquamous carcinoma (ASC) is a rare, aggressive subtype of non-small cell lung cancer (NSCLC), characterized by both adenocarcinoma (ACC) and squamous cell carcinoma (SCCC) components. Due to its rarity, the clonal origin, evolutionary drivers, and tumor microenvironment (TME) of ASC remain poorly understood.

We conducted clinical and prognostic analyses on 76 ASC patients. Nine patients with available tissues underwent microdissection to isolate matched ACC and SCCC regions, which were then analyzed using whole-exome sequencing (WES), copy-number analysis, phylogenetic reconstruction, and multiplex immunofluorescence (mIF) for immune profiling.

Multivariate Cox regression identified the histological subtype of the ACC component as an independent prognostic factor, with solid and micropapillary subtypes associated with the poorest overall survival. WES revealed frequent mutations in TP53 (56%), EGFR (33%), MET (33%), and PIK3CA (22%) across tumor regions. Shared truncal mutations and copy-number alterations between ACC and SCCC regions suggest a common monoclonal origin. Smokers had a significantly higher tumor mutational and neoantigen load than non-smokers, with greater overlap in mutations between ACC and SCCC. Phylogenetic analysis showed distinct evolutionary patterns: smokers' tumors displayed smoking-related and APOBEC mutational signatures, while non-smokers had aging-related signatures. mIF analysis revealed significantly higher immune cell infiltration (CD8⁺ T cells, CD4⁺ T cells, B cells, macrophages, CAFs) in ACC regions compared to SCCC.

Our findings support a monoclonal origin for ASC, with smoking influencing divergent evolutionary trajectories and immune microenvironment characteristics between ACC and SCCC. These insights provide a molecular framework for personalized ASC therapies.