Prognostic significance and tumor cell interactions of tumor-associated neutrophils in gastric cancer

BACKGROUND: This study aimed to determine the clinicopathologic implications and tumor cell interaction of CD66b-positive (CD66b +) tumor-associated neutrophils (TANs) and CD66b and CD177 double positive (CD66b + /CD177 +) activated TANs in gastric cancer (GC). METHODS: Single immunohistochemistry (sIHC) for CD66b, CD8, HER2, p53, and E-cadherin was performed in 1,060 patients with GC, together with microsatellite instability (MSI) testing and Epstein-Barr virus (EBV) in situ hybridization. Opal multiplex immunofluorescence (mIF) for cytokeratin (CK), CD66b, and CD177, and sIHC for ERα was performed on 59 microsatellite-stable (MSS) and 60 MSI-high (MSI-H) GCs. The immune cell densities were quantified using QuPath for sIHC and InForm for mIF. RESULTS: On sIHC, a high density of CD66b + TANs was correlated with HER2 positivity, EBV positivity, and MSI-H phenotype (p < 0.001). Combined analysis of CD66b + TAN densities in the tumor center and invasive margin showed a significant association with better OS (p = 0.036), independent of major clinicopathologic factors. TAN densities were significantly associated with OS in female patients (p < 0.001), but not in male patients (p = 0.574). The density of ERα-positive immune cells was inversely correlated with TAN density (ρ = -0.41, p < 0.001). mIF analysis demonstrated that both CD66b + TANs and CD66b + /CD177 + activated TANs were significantly more abundant in MSI-H GC than in MSS GC. Moreover, the distance between TANs and CK + GC cells was significantly shorter in MSI-H GC than in MSS GC. A shorter distance between CD66b + /CD177 + activated TANs and GC cells was also associated with a better OS (p = 0.041). CONCLUSION: A high TAN density in GC is associated with a favorable prognosis, and the spatial proximity of TANs to GC cells, especially in MSI-H GC, provides additional prognostic value. These findings provide basic knowledge for the development of therapeutic targets related to TANs in GC.