Proof-of-principle 4-marker spatial profiling reveals distinct, location-independent immune clusters in biliary tract cancers

Biliary tract cancers (BTCs) are aggressive malignancies with limited treatment options. Despite advances with durvalumab plus chemotherapy, prognosis remains poor. The tumor immune microenvironment (TIME) is increasingly recognized as a determinant of immunotherapy response, yet its spatial organization in BTC is poorly understood. We applied multiplex immunofluorescence to pre-treatment tumor samples from 11 patients with advanced BTC, staining for CD4, CD8, CD20, and CD163 to identify T cells, B cells, and macrophages. Across 198 regions of interest sampled at the invasive tumor margin, approximately 400,000 individual cells were segmented and phenotyped. Spatial organization was analyzed using hierarchical clustering, distance-based approaches, and cellular neighborhood classification. Unsupervised analysis identified two distinct immune phenotypes, one enriched in CD4 + T cells and CD163 + macrophages and the other in CD8 + T cells and CD20 + B cells. These phenotypes displayed distinct spatial architectures, with CD4 + T cells and CD20 + B cells acting as organizing hubs, respectively. Targeted sequencing, available for a subset of cases, showed a higher frequency of co-occurring TP53 and ARID1A alterations in the B-cell–enriched phenotype. No statistically significant differences in overall or progression-free survival were observed between immune phenotypes. However, among patients treated with adjuvant cisplatin/gemcitabine alone, lower CD20/CD8 fractions and higher CD163 abundance at baseline were associated with improved survival. Spatial immune profiling revealed distinct BTC immune architectures at the invasive margin, with different possible impact on survival, thus warranting further characterization and validation in larger cohorts.