Spatial proteomics deciphers the immune niche landscape of non-ampullary duodenal adenocarcinoma

Abstract

Non-ampullary duodenal adenocarcinoma (NADA) is a rare malignancy with an incompletely characterised tumour immune microenvironment (TME). We aimed to elucidate its immune niche landscape and validate a clinically applicable transcriptomic surrogate. In 38 resected NADA cases, multiplex immunofluorescence spatial proteomics was performed on tissue microarrays, validated by cohort-wide whole-slide imaging. A 10-gene qRT-PCR panel capturing adaptive immune resistance mechanisms was integrated with spatial data. Molecular microsatellite instability (MSI) testing confirmed mismatch repair (MMR) status in all integrated cases. Spatial proteomics identified seven distinct cellular niches; the immune-active “Hot spot” niche showed a trend toward prolonged survival. The 10-gene panel classified patients into “Hot,” “Intermediate,” and “Cold” clusters, with the “Hot” cluster significantly enriched in “Hot spot.” A pMMR subgroup with abundant “Hot spot” (7/29, 24.1%) was identified, most belonging to the “Hot” cluster (5/7, 71.4%). Molecular MSI testing confirmed all pMMR “Hot” cases as microsatellite stable. External transcriptomic analysis revealed enrichment of interferon-gamma and inflammatory response pathways in the “Hot” group. Spatial proteomics identified an immune-active “Hot spot” niche in NADA. The 10-gene panel serves as a spatial surrogate, identifying a microsatellite-stable pMMR subgroup that may represent a biologically relevant subgroup for future investigation.