Spatial organisation of tumor-infiltrating B cells in Claudin 18.2-expressing advanced gastric cancer predicts response to immune checkpoint inhibition

Background : Claudin 18.2 (CLDN18.2), a tight junction protein aberrantly expressed during malignant transformation of gastric cancer (GC), has emerged as a key therapeutic target. This study seeks to understand the role of CLDN18.2 in shaping the tumor microenvironment (TME). Methods : This single-center study included 99 patients with advanced gastric cancer treated first-line immune checkpoint inhibition (ICI) agent(s) with chemotherapy. CLDN18.2 expression was determined using immunohistochemistry (VENTANA 43-14A, Roche) on FFPE biopsies, reported as % 2+/3+. To enable a balanced representation of CLDN18.2 status, a median split approach was employed. Samples were also stained with multiplex-IHC (CK, CD4, CD8, FOXP3, CD68 and CD20) to elucidate the corresponding TME. Orthogonal validation was undertaken through independent analyses of whole transcriptome sequencing (WTS) data from 6 cohorts comprising 1,098 GC tumor samples. Cellular neighbourhood (CN) analyses were conducted using a k -nearest neighbors' algorithm. Survival analyses were conducted with a Cox-proportional hazards model. Results : We retrieved CLDN18.2 status through median-split of CLDN18.2 expression (25.1%), yielding 44 CLDN18.2 high samples and 45 CLDN18.2 low samples. We observed an increase in CD20 + B cell density in CLDN18.2 high samples (p=0.042), whereas no significant differences in cell density were observed in CD4 + /CD8 + T cells, CD68 + macrophages and FOXP3 + Tregs. These findings were orthogonally validated utilizing immune cell deconvolution (xCell, Epic & MCP-counter) across the 6 independent WTS cohorts. CLDN18.2 status alone did not predict for response or survival (overall survival [OS], HR=1.02, p=0.947). GCs with increased CD20 + B cell density was found to confer a greater magnitude of benefit from first-line ICI (OS, HR=0.71, p=0.196), but the benefit was largely conserved amongst CLDN18.2 low tumors and not in CLDN18.2 high tumors (OS interaction p=0.109). We identified 4 B cell related CNs: B-TumorInfiltrating, B-MacrophageNiche, B-LA/TLS (Lymphoid Aggregate/Tertiary Lymphoid Structure) and B-ImmuneNiche. In CLDN18.2 high tumors, we observed a relative increase in CD20 + B cell abundance within B-TumorInfiltrating and B-MacrophageNiche CNs and a relative decrease in B cell abundance within B-LA/TLS and B-ImmuneNiche CNs in comparison to CLDN18.2 low tumors. While B-ImmuneNiche and B-LA/TLS conferred ICI sensitivity, the relative enrichment of B cells within a B-TumorInfiltrating CN compared to a B-ImmuneNiche CN conferred ICI resistance, particularly in CLDN18.2 high tumors (OS, HR=2.60, p=0.040). Conclusions : The CLDN18.2 high gastric cancer microenvironment is characterized by an enriched humoral response. Intra-tumoral B cell infiltration confers ICI resistance in a CLDN18.2 high microenvironment.