Single cell and spatially resolved determinants of the clinical outcome of patients (pts) treated with locally advanced/metastatic head and neck squamous cell carcinoma (LA/M SCCHN) treated with immunotherapy (IO)

Background: We developed a composite Gene Expression Signature (cGES) comprising 22 protective (P) and 19 adverse (A) genes, agnostic of the tumor type, stratifying IO-treated pts in 3 risk groups : low (L-), intermediate (I-) and high-risk (H-) (abstract#6360 AACR2025). Herein, we validate its predictive value for progression free (PFS) and overall survival (OS) in a prospective cohort and uncover the single-cell and spatial determinants underlying clinical outcomes. Methods: (1) The cGES was computed in 170 pts with LA/M SCCHN from 2 trials (NCT03226756; NCT03412058). (2) Next, we built a scRNAseq SCCHN atlas (scAt) of 219,138 cells from 77 independant pts including 19 and 21 pts classified as H-risk and L-risk respectively, and studied the distribution and functional states of cell populations between the 2 groups of pts. Cell-type-specific enrichments of A & P genes werequantified and intercellular communication networks were inferred to identify ligand-receptor (L-R) interactions. (3) Deconvoluted Visium data from 12 of the 77 SCCHN including 3 H-risk and 3 L-risk pts were used to map the spatial organization of cGES and to localize specific L-R interactions. Results: (1) Multivariate analysis adjusted for ECOG, age, gender, and alcohol/tobacco use showed that I- and L-risk pts had significantly better outcomes. For PFS, hazard ratios were 0.64 (p=0.04) and 0.51 (p=0.005), and for OS 0.81 (p>0.05) and 0.54 (p=0.015), respectively. (2) Analysis of the scAt revealed that A genes from cGES were mainly expressed by epithelial and stromal cells. Conversely, P genes were mainly expressed by immune cells. H-risk tumors exhibited an altered communication landscape marked by strengthened epithelial junctions, developmental signaling, and universally increased EGFR L-R activity, whereas L-risk tumors showed an immune-enriched network dominated by extracellular remodeling, adaptive immune activation and immune cell recruitment. (3) Visium deconvolution identified 5 major cell-composition clusters (C), with epithelial-enriched C2 showing the highest A genes expression, while T cell-enriched C4/C5 displayed the strongest P genes expression. H-risk tumors exhibited prominent EGFR ligand expression, consistent with the results from the scAt data. In contrast, L-risk tumors displayed increased antigen-presentation, T-cell chemotaxis, and complement signaling. Conclusion: cGES reliably stratified prognosis and reflects tumor ecosystem biology. H-risk tumors were dominated by epithelial-driven, EGFR-centered signaling, whereas L-risk tumors showed coordinated inflammatory T-cell-oriented programs. These findings suggest cGES as a prognostic and mechanistic biomarker, and provides a rational for EGFR-IO combination strategies to improve outcomes in LA/M SCCHN.