Microsatellite stable (MSS) metastatic colorectal cancer (mCRC) remains poorly sensitive to immune checkpoint blockade, highlighting the need for improved biological stratification and novel combination strategies. The AVETUXIRI trial evaluated avelumab (anti–PD-L1), cetuximab (anti-EGFR), and irinotecan in heavily pretreated MSS mCRC patients. Here, we report baseline biological features associated with treatment benefit in metastatic biopsies.

55 MSS chemo-refractory mCRC patients (28 RAS wild-type, 27 RAS mutated; anti-EGFR–refractory if RAS wild-type) received cetuximab, irinotecan, and avelumab. Multi-layer immune and metabolomic profiling was performed on baseline tumor biopsies and matched blood samples, including RNA sequencing and multiplex immunofluorescence (mIF) in tumors, and flow cytometry, mass spectrometry, and TCR sequencing in blood. Based on previously reported clinical outcomes, patients were stratified according to treatment benefit using progression-free survival (≥6 vs <6 months), overall survival (≥12 vs <12 months), and best overall response (tumor shrinkage vs growth).

Transcriptomic analyses revealed enrichment of immune-related signatures in patients with treatment benefit, including higher immunoediting scores, correlating with improved progression-free and overall survival. Spatial profiling by mIF confirmed that baseline CD3+ and CD8+ T-cell densities, particularly with close tumor proximity, were strongly associated with outcomes. Beyond T cells, immune deconvolution identified B cells as key discriminators between benefit groups, validated by mIF with increased intratumoral CD20+ B-cell infiltration in patients with treatment benefit, whereas others showed higher IgG deposition, suggesting distinct humoral dynamics. In contrast, circulating immune profiling showed limited concordance with tissue findings, although baseline TH17 cells were associated with outcomes, highlighting the challenge of translating spatial immune signatures into liquid biopsy biomarkers. Finally, non-benefit patients exhibited enrichment of metabolic pathways, including fatty acid and retinol metabolism, suggesting a link between metabolic rewiring and immune resistance.

Baseline immune and metabolomic contexture shapes clinical benefit from anti–PD-L1–based therapy in MSS mCRC. Treatment benefit was associated with enhanced immune activation whereas lack of benefit was characterized by metabolic pathway enrichment. Integrative spatial, transcriptomic, and metabolic profiling may guide biomarker-driven combination immunotherapy strategies in MSS mCRC.

Supported by a grant from the National Fund for Scientific Research [Télévie/FNRS 7.4600.23]. This academic study (investigor sponsorized trial) received a fixed grant and supply for the study drugs from Merck.