Intratumoral effector T-cells and macrophages underlie prolonged survival after PD-1 axis blockade in metastatic non-small cell lung cancer (mNSCLC)

Background: Although PD-1 axis inhibitors have improved outcomes in patients with mNSCLC, only a subset of patients derives long-term benefit. Most studies assessing determinants of sensitivity have relied on short-term endpoints such as response, potentially overlooking biological features underlying durable tumor control. We hypothesized that patients achieving prolonged response after PD-1 axis blockade exhibit distinct tumor microenvironment (TME) features compared with those with primary resistance or limited response. Methods: We assembled a retrospective cohort of bulk whole-transcriptome data from 342 pretreatment tumor samples from patients with mNSCLC treated with PD-1 axis blockade alone or in combination with anti-CTLA-4 therapy from Yale (n=31), Stand Up To Cancer (n=73), and Samsung Medical Center (n=278). Patients were classified as having primary progression (PP; PFS <3 mo; n=200), non-exceptional response (NER; PFS 6-36 mo; n=116), and prolonged survival (PS; PFS >36 mo; n=26). After batch correction, differential expression (limma) and CIBERSORT were used to identify genes and immune cells associated with PS. Single-cell RNA sequencing from a publicly-available lung cancer immune cell atlas (n=234) was used to refine immune cell signatures and assess their association with outcomes via ssGSEA. Spatial proteomics (Phenocycler-Fusion) performed on baseline samples from patients treated with PD-1 axis blockade (n=55; PP=21, NER=26, PS=8) at Yale and the University of Queensland was used for validation. Results: Tumors from PP, NER, and PS demonstrated a stepwise increase in HLA class I antigen-presentation machinery (APM) transcripts (PSMB9, ERAP2), memory T-cell markers (CXCR6), and CD8⁺ T-cell infiltration. PD-L1 expression was markedly higher in NER than PP, but similar between NER and PS. The most significantly enriched genes in PS relative to NER were macrophage-related, with higher absolute M1 (Cliff's delta=0.34, p=0.007) and M2 (delta=0.29, p=0.023) tumor-associated macrophage (TAM) scores. This difference was not observed between NER and PP. Four percent of patients with low (≤ median) calculated CD8⁺ T-cell and TAM levels were PS, compared with 15% of those with high CD8⁺/TAM levels (odds ratio=4.5, p=0.0083). Single-cell analysis identified 15 TAM subsets, several enriched in PS but none in PP. Spatial proteomics confirmed an increase in the proportion of stromal TAMs in PS (PS vs NER: delta=0.62, p=0.0079; PS vs PP: delta=0.58, p=0.016) and revealed a stepwise decrease in the proportion SMA⁺ fibroblasts across clinical benefit groups. Conclusion: Prolonged survival after PD-1 blockade in mNSCLC is associated with distinct TME features including increased expression of APM components, and increased CD8 T-cells and TAMs. These insights may inform the development of predictive biomarkers and novel therapeutic strategies.