Introduction Gastric-type mucinous carcinoma (GAS) is the most common subtype in HPV-unrelated endocervical adenocarcinoma. GAS has aggressive behavior and worse clinical outcome compared with usual-type endocervical adenocarcinoma (UEA). In this research, we delineated the tumor immune microenvironment of GAS by comparing with UEA and found the adverse effect of TLS in them.

Methods Multiplex immunofluorescence (mIF) and RNA sequencing were applied in this study. We optimized 4 mIF panels about tumor-infiltrating immune cell, the subtypes of macrophages, tertiary lymphoid structure (TLS) and regular T cell (Treg) and regular B cell (Breg)

Results By analyzing mIF data, GAS tumors had less infiltration of CD4⁺ T cells and CD8⁺ T cells but more macrophages than UEA. In GAS patients, macrophage infiltration was significantly increased in patients with positive lymph node metastases and advanced stage, especially the M2 type macrophages. Functional pathway enrichment analysis also shew the immune-activated related pathways were more enriched in UEA tumors. By statistics, approximately 15% of patients had mature TLS. Surprisingly, mature TLS formation was associated with shorter overall survival time in both GAS and UEA. Patients with mature TLS had more Tregs and Bregs infiltrating in both the tumor region and the lymphocyte aggregation region. Gene expression analysis shew the tumor cell proliferation was increased, while the immune-activating was reduced in tumors with mature TLS.

Conclusion/Implications We found GAS had less lymphocytes infiltration than UEA, but more macrophage infiltration, especially M2-type macrophages. And the formation of mature TLS was a crucial adverse prognostic factor in GAS and UEA.