Abstract 798: Tumor-associated neutrophil density and cancer cell interaction in microsatellite stable and unstable gastric cancer using multiplex immunofluorescence.

Abstract Purpose: Tumor-associated neutrophils (TANs) play diverse roles in the tumor microenvironment, yet their clinicopathologic and prognostic significance in gastric cancer (GC), particularly the relevance of activated TAN subsets and their spatial interactions with tumor cells, remains unclear. This study aimed to characterize TAN density, activation status, and spatial proximity to tumor cells, with focus on microsatellite instability-high (MSI-H) GC. Methods: Single immunohistochemistry (sIHC) for CD66b, CD8, HER2, p53 and E-cadherin was performed in 1,060 GCs, together with MSI testing and Epstein-Barr virus (EBV) in situ hybridization. To explore potential sex-related differences in TAN-associated survival, ER sIHC was additionally performed. To define functional TAN subsets, Opal multiplex immunofluorescence (mIF) for cytokeratin (CK), CD66b, and CD177 was conducted in 59 microsatellite-stable (MSS) and 60 MSI-H cases. Immune cell densities were quantified using QuPath (sIHC) and InForm (mIF). Spatial proximity between TANs and CK+ tumor cells was calculated. Survival was assessed by Kaplan-Meier analysis. Results: A high density of CD66b+ TANs on sIHC was more frequently observed in intestinal-type GC and at lower pT stages (P0.001). A higher TAN density was correlated with HER2 positivity, membranous E-cadherin expression, EBV positivity, and the MSI-H phenotype (P0.001). A moderate correlation was found in TAN densities between the tumor center and invasive margin. Patients with a high density of CD66b+ TANs tended to have better overall survival (OS), and combined analysis of central and invasive margin TAN densities indicated an independent prognostic factor (P=0.036). In subgroup analysis, higher TAN density was associated with improved OS in female patients (p0.001). By mIF, the densities of CD66b+ TANs and CD66b+/CD177+ activated TANs were significantly higher in MSI-H than MSS GC (p0.01). The distance from GC cell to the nearest CD66b+ TAN or CD66b+/CD177+ activated TAN was significantly shorter in MSI-H GC (p0.001). TAN density was higher in areas adjacent to tumor cells and lower in distant regions, with a more pronounced gradient in MSI-H GC. A shorter TAN-tumor distance was associated with better survival. In particular, closer proximity of activated CD66b+/CD177+ TANs to tumor cells was independently associated with improved OS (p=0.041). Conclusions: The distance between TANs and GC cells was significantly shorter in MSI-H tumors, and a shorter TAN-tumor distance-especially for activated CD66b+/CD177+ TANs-was associated with better overall survival. These findings highlight TAN-tumor spatial proximity as a key prognostic factor and a potential target for future therapeutic strategies in GC. Citation Format: Soo Kyung Nam, Seo Hwan Yu, Sun Young Ahn, Yujun Park, Yoonjin Kwak, Sujin Oh, Kyoung Un Park, Hye Seung Lee. Tumor-associated neutrophil density and cancer cell interaction in microsatellite stable and unstable gastric cancer using multiplex immunofluorescence abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 798.