Tumor microenvironment of ampulla of Vater cancer reveals distinct spatial niche of CAF, TIL, and TAM and their correlation with clinicopathologic features

Background: While the individual roles of cancer-associated fibroblasts (CAFs), tumor-infiltrating lymphocytes (TILs), and tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) of ampulla of Vater (AoV) carcinomas are recognized, a detailed understanding of their collective interplay and its correlation with clinicopathologic features and patient survival is still lacking. Methods: A retrospective cohort of 87 patients with resected stage IB-III AoV adenocarcinoma (Seoul St. Mary's Hospital between 2007 and 2021) was selected. TMAs (2-mm cores) were constructed and immunostained for tumor cell markers (EGFR, HER2, and c-MET), TME markers (CD3, CD8, FOXP3, CD68, CD163, alpha-SMA, FAP, PDPN, and POSTN), and Masson's Trichrome (MT) staining for fibrosis. Multiplex IF was performed for high-plex spatial proteomic analysis (CellScape). All slides were digitized and analyzed to quantify the immune cell densities (cells/mm2), mesenchymal marker H-scores, and spatial relationships (cell-cell distance) to define the TME niches. The primary endpoints analyzed using R were disease-free survival (DFS) and overall survival (OS). A two-sided p-value of <0.05 was considered to be statistically significant. Results: TAM markers showed a significant positive correlation with T-cell markers but a negative association with CAF markers. FAP and POSTN were positively correlated with each other but negatively correlated with PDPN and IL6. ACTA2 and IL6 were negatively correlated with most other markers, while being positively correlated with each other. Univariate Cox regression for DFS identified high EGFR (HR=6.89, 95% CI: 2.94-16.20, p=0.026), FAP (HR=2.21, 95% CI: 1.27-3.86, p=0.027), and high fibrosis (MT) (HR=2.49, 95% CI: 1.42-4.37, p=0.008) as unfavorable prognostic factors. CD3 and POSTN showed a trend towards prognostic significance for OS (p=0.07 and p=0.06, respectively). In multiplex IF, spatial analysis revealed that TAMs (CD68 and CD163) were located closest to the tumor cells, followed sequentially by CAF markers (vimentin and SMA), Collagen IV, and TIL markers, which were the most distant. Conclusions: Our findings demonstrate that TME components in AoV carcinoma form distinct spatial niches, characterized by TAMs proximal to tumor cells and TILs in the distal region. This specific architecture, especially when dominated by high FAP and fibrosis, is significantly associated with poor survival, highlighting the importance of spatial TME analysis as a critical prognostic tool.