Potential effects of TGF-β inhibition on immune resistance by targeting immunosuppressive microenvironment in advanced gastric cancer (AGC): Multi-omics post-hoc analysis of the K-Umbrella-06 trial.

2612 Background: TGF-β signaling promotes immune exclusion by driving fibrosis and hindering T-cell infiltration. We performed multi-omics post-hoc analyses of K-Umbrella-06 to identify mechanisms and predictive biomarkers for TGF-β/PD-L1 dual blockade in HER2-negative AGC. Methods: K-Umbrella-06 (NCT04835896) was an investigator-initiated, single-arm, phase Ib/II study enrolling HER2-negative AGC pts who had progressed on 1st-line treatment. Pts received bintrafusp alfa (TGF-β trap/anti–PD-L1) and weekly paclitaxel. Pretreatment H&E whole-slide images (WSIs) were analyzed with an AI-powered WSI analyzer to quantify fibroblast and endothelial cell (EC) densities and immune phenotypes. A separate cohort treated with nivolumab + paclitaxel (n=26) served as a control. Integration of baseline tissue transcriptomics and serial ctDNA sequencing (baseline, 1 st RECIST assessment, and progression) was performed to identify predictive biomarkers and resistance mechanisms. Results: At a median follow-up of 46 months in 30 enrolled patients, the mPFS and mOS were 3.8 and 8.9 months, respectively; notably, 5 patients achieved durable responses with PFS exceeding 3 years. AI-WSI analysis revealed that high fibroblast density (≥median) was a significant predictor of superior outcomes (mPFS: 4.3 vs. 2.0 months, HR 0.29, P=0.007; mOS: 20.4 vs. 5.8 months, HR 0.28, P=0.012). Conversely, patients with higher intratumoral EC density had shorter mPFS (2.0 vs. 5.9 months, HR 2.29, P=0.058) and mOS (4.0 vs. 8.9 months, HR 1.61, P=0.285). Notably, patients with the immune-excluded phenotype achieved a higher ORR (60.0% vs. 18.2%) and prolonged survival compared to inflamed/desert types. These biomarkers were not predictive in the nivolumab + paclitaxel cohort, suggesting a TGF-β specific effect. Transcriptomic profiling of responders showed enrichment in TGF-β signaling (normalized enrichment score [NES] 2.87, P<0.001) and SMAD2/3 activity (NES 2.99, P<0.001), alongside increased naive CD8+ T cells and inflamed EC cells. Conversely, non-responders exhibited higher M2 macrophage and abundant angiogenic tip cells. Serial ctDNA analysis identified emergent TGF-β pathway alterations as a potential mechanism of acquired resistance (OR 7.30, P=0.08). Conclusions: Integrated AI-WSI, transcriptomic, and longitudinal ctDNA analyses suggest that TGF-β pathway activation and an immune-excluded phenotype are associated with clinical benefit from bintrafusp alfa plus paclitaxel, and may identify a responder subset distinct from conventional PD-1/PD-L1–based therapy. These findings support further evaluation of anti–TGF-β strategies for AGC with an immunosuppressive tumor microenvironment. Clinical trial information: NCT04835896 .