Multi-modal molecular profiling of CDH1 and CLDN18.2 to identify predictive biomarkers for zolbetuximab efficacy in patients with advanced gastric adenocarcinoma: The ZOL-C3 study.

3148 Background: Zolbetuximab is a chimeric monoclonal antibody designed to bind to claudin 18.2 (CLDN18.2) and induce antibody-dependent cell-mediated cytotoxicity (ADCC). Although phase 3 trials established its efficacy in CLDN18.2-positive advanced gastric adenocarcinoma (GAC), robust predictive biomarkers beyond protein expression remain an unmet need. We investigated how epithelial-mesenchymal transition (EMT), mediated by CDH1 alterations and the tumor mutational burden (TMB), modulates the therapeutic target and affects zolbetuximab-mediated ADCC efficacy. Methods: This study employed a multi-layered integrative framework across four independent datasets to cross-validate genomic, transcriptomic, and phenotypic findings: (1) mechanistic characterization using the TCGA-STAD cohort (n=375); (2) the genomic screening of 2,483 patients with GAC in the Japanese national database (C-CAT), which identified 49 treated with zolbetuximab-containing regimens; (3) a spatial transcriptomic evaluation (10× Genomics Visium platform, GSE251950); and (4) immunohistochemical (IHC) validation of CLDN18.2 in 36 institutional GAC patients. The trial is registered at UMIN-CTR (UMIN000060223), a WHO ICTRP primary registry. Results: In the TCGA cohort, CDH1 low-expression was significantly associated with 3.2-fold lower CLDN18 mRNA levels than in the high-expression group (p <0.001). CDH1 expression was strictly regulated by promoter methylation (p <0.001), whereas CLDN18 down-regulation in CDH1-low tumors appeared to be driven by non-epigenetic EMT pathways. Spatial transcriptomics revealed a spatial correlation between CDH1 and CLDN18 mRNAs within the malignant epithelial compartment (p <0.001). The objective response rate within the zolbetuximab-treated C-CAT subset was 22.2%. TMB was markedly lower in responders than in non-responders (median 1.82 vs. 4.0 mut/Mb; p = 0.050). Furthermore, patients without CDH1 single nucleotide variants had higher ORR (p = 0.086). Institutional IHC validation revealed a superior response in cases that retained membranous CDH1 expression to that in cases with the loss of epithelial integrity (100% vs 50% ORR; p=0.088). Conclusions: These results demonstrate that CDH1/E-cadherin integrity and low TMB are critical co-determinants of CLDN18.2 expression and zolbetuximab efficacy. Maintenance of the epithelial phenotype appears to be essential for optimal zolbetuximab-mediated ADCC. The integration of comprehensive molecular profiling, including the CDH1 status and TMB, may significantly refine patient selection, with a shift from simple target positivity to a more biological enrichment strategy for zolbetuximab therapy.