Use of tumor-intrinsic pathways to identify novel targets in immune-cold esophagogastric adenocarcinoma.

451 Background: In first-line E/GEJ/G adenocarcinoma, anti-PD-1 + chemotherapy improves outcomes, yet many patients do not respond. Beyond PD-L1 CPS, tumor-intrinsic biomarkers of primary resistance remain limited. We applied a validated IFN-γ–linked T cell-inflamed (Tinfl) framework to define exclusionary programs and nominate targets. Methods: Bulk RNAseq was analyzed from TCGA ESCA/STAD adenocarcinoma (n=79, discovery) and a de novo Mayo cohort of pretreatment samples (n=74, validation) with harmonized survival and response data. Tinfl scores (160-gene signature) defined quartiles (Q1 cold, Q4 hot). Cold versus hot tumors were compared by edgeR + limma-voom to identify differentially expressed genes (DEGs) (Bioconductor v3.20). Pathway and upstream-regulator/causal-network analyses used Ingenuity Pathway Analysis seeded by top DEGs (|log2FC|>1.5, p<0.05). Survival was assessed by Cox proportional hazards models (two-sided α=0.05) and KM log-rank testing (α=0.05). A 14-gene Tinfl-weighted cold signature produced a per-sample composite score. Results: In the Mayo cohort, median age was 62 years; 72% were stage IV at diagnosis; 24% were HER2-positive and 3% MSI-H; primary sites were GEJ/esophagus/gastric 50%/34%/16%; PD-L1 CPS <1/1–5/6–49/≥50 occurred in 18%/46%/27%/4%; patients received a mean of 2.4 lines of therapy. Across discovery and validation cohorts, Tinfl-cold tumors showed significantly higher expression of developmental and lineage transcription factors (HOXC12, HOXC13, ZIC1, DLX3, EVX1), an oncofetal post-transcriptional hub (IGF2BP1), cancer-testis and piRNA components (MAGEA4, TDRD12, THEG), and neuronal or transport genes (GABRQ, KCNF1, LDLRAD1, SLC6A10P) (logFC >2, p<0.05). Pathway analysis highlighted chromatin and lineage regulators (KAT6A, SIRT1, LEF1), TGF-β signaling (TGFBR2), and repressors of innate DNA/IFN sensing (TREX1, IRF2BP2) (Z-score >2, p<0.05). The 14-gene Tinfl-weighted cold signature was higher in cold versus hot tumors (p<0.05) and showed a directional association with inferior overall survival in the Mayo validation cohort. Among patients who received first-line anti-PD-1–containing regimens, several signature members and the composite score were higher in non-responders (p<0.05). Conclusions: Using a discovery–validation design, we define tumor-intrinsic expression pathways that characterize Tinfl-cold E/GEJ/G adenocarcinoma. We also derive a concise 14-gene Tinfl-weighted cold signature that is enriched in non-responders and associated with inferior overall survival. Targetable nodes such as IGF2BP1 and MAGEA4, together with chromatin and Wnt/TGF-β regulators, support rational combinations with PD-1 blockade. Planned validation in a third cohort, single-cell confirmation of tumor-cell expression, and in vivo cytokine profiling in knockdown models will refine mechanisms and nominate clinical candidates.