Transcriptome differences revealed by high-plex digital spatial profiling associated with endocrine sensitivity for hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) early breast cancer (EBC)

Objective: This study aims to explore the spatial and transcriptome differences associated with endocrine sensitivity in HR+/HER2- EBC.

Methods: Postoperative FFPE samples of patients with HR+/HER2- EBC were collected. All patients received adjuvant tamoxifen after surgery. The follow-up period was ten years. Based on disease-free survival, patients were stratified into the ET-resistant and ET-sensitive groups. A total of 111 spatially resolved regions in three tissue compartments defined by morphology markers [tumor (PANCK+), leucocytes (CD45+), and nonimmune stroma (CD45-/PANCK-)] were assessed based on GeoMx digital spatial profiling. ET-associated genes were collected from over 5,000 published articles. Enrichment analyses were performed using multiple bioinformatics approaches.

Results: A specific panel comprising 235 ET-related genes was constructed. In PANCK+ regions, 11 upregulated and 7 downregulated differentially expressed genes (DEGs) were identified when comparing the ET-resistant with the ET-sensitive group, with GATA3 showing the most differential expression. A total of 44 KEGG pathways were enriched, among which Th1 and Th2 cell differentiation had the greatest significance (p=6.25E-4). GO functions were mainly enriched in the nucleus, nucleoplasm, and negative regulation of transcription. In CD45+ regions, 14 upregulated DEGs were found, with most enriched in the HER signaling pathway (p=5.57E-08). Cellular activities, including protein serine/threonine kinase activity, protein phosphorylation, and protein kinase activity, were profoundly enriched. In CD45-/PANCK- regions, 21 upregulated and 6 downregulated DEGs were detected in the ET-resistant group versus the ET-sensitive group. Proteoglycans in cancer (p=3.98E-07) and signaling pathways regulating the pluripotency of stem cells (p=8.70E-07) showed higher statistical significance. GO functions were mainly enriched in the nucleus, nucleoplasm, and positive regulation of transcription by RNA polymerase II.

Conclusion: Our study first shows transcriptome heterogeneity among patients with different responses to ET across three PANCK+ (CD45+ and CD45-/PANCK-) regions. This may provide a reference for exploring molecular mechanisms of endocrine resistance.