Cellular, chromatin-accessibility, and spatial contextualization of bulk transcriptional patterns linked to positive peritoneal cytology in endometrial cancer.

BACKGROUND

Positive peritoneal cytology (PPC) identifies a clinically adverse subset of endometrial cancer, but the tumor transcriptional patterns accompanying PPC and their cellular origins remain incompletely resolved. We evaluated PPC-linked bulk RNA patterns in TCGA-UCEC and contextualized the resulting programs using independent single-cell, chromatin-accessibility, and spatial datasets.

METHODS

TCGA-UCEC clinical BCR XML, primary-tumor RNA sequencing, molecular annotations, ABSOLUTE purity, and TCGA Clinical Data Resource outcomes were integrated at the patient level. The final cohort contained 408 tumors with explicit PPC status. Prespecified ranked gene-set enrichment, covariate-adjusted models, multiple imputation, and survival analyses were performed. Frozen programs were mapped in GSE173682 paired scRNA-seq/scATAC-seq and GSE203612 scRNA-seq/Visium data. Seven commercial human cell models obtained from the American Type Culture Collection were used for qRT-PCR localization of representative Program A and Program B transcripts.

RESULTS

The cohort included 351 PPC-negative and 57 PPC-positive tumors. No individual gene reached genome-wide BH-FDR<0.05. Ranked analysis showed relative enrichment of epithelial-mesenchymal transition (EMT), hypoxia, TGF-beta signaling, and angiogenesis in PPC-positive tumors, with relative depletion of cytotoxic-lymphocyte, T-cell-inflamed, and antigen-presentation programs. PPC remained associated with shorter progression-free interval (PFI) (HR = 2.77, 95% CI = 1.73-4.43), overall survival (OS) (HR = 2.84, 95% CI = 1.66-4.84), and disease-specific survival (DSS) (HR = 3.47, 95% CI = 1.86-6.46). Program A showed a replicated broad stromal-vascular source, whereas Program B localized to NK/CD8 compartments. After cell-type and patient adjustment, only Program B retained clear RNA-ATAC concordance. Program A was strongly spatially organized in one UCEC section (Moran's I = 0.769; FDR = 0.0015), but composition-adjusted analyses did not support cytotoxic exclusion or stable hypoxia/TGF-beta coupling. qRT-PCR showed higher Program A scores in stromal/perivascular models than in tumor-epithelial models (mean difference 1.52 standardized units, 95% CI = 1.34-1.71; P < 0.001) and higher Program B scores in NK than in non-cytotoxic models (mean difference 2.09, 95% CI = 1.97-2.22; P < 0.001). Tumor CD274 transcript abundance was not higher in PPC-positive tumors and did not credibly modify PPC-associated risk.

CONCLUSION

PPC-positive endometrial cancers exhibit coordinated stromal/mesenchymal-high and cytotoxic/antigen-presentation-low ranked bulk transcriptional directions. Single-cell, chromatin-accessibility, spatial, and qRT-PCR data consistently localize the dominant programs to stromal-vascular and cytotoxic-lymphocyte compartments. PPC remains a strong adverse clinical marker, whereas tumor CD274 transcript abundance does not explain its prognostic association.