Exploring Intratumoral Heterogeneity in Mixed Neuroendocrine-Nonneuroendocrine Neoplasms with Spatial Transcriptomics: Even More Diverse Than Anticipated

Abstract Mixed neuroendocrine–nonneuroendocrine neoplasms (MiNEN) are usually highly aggressive tumors characterized by marked histological heterogeneity, most commonly represented by mixed adenocarcinoma and poorly differentiated neuroendocrine carcinoma (NEC). However, beyond morphological observations, the biological basis and implications of this heterogeneity remain incompletely understood. In this study, we combined component-specific next-generation sequencing and spatial transcriptomics to investigate three mixed adenocarcinoma-NEC cases from different anatomical sites (ileocecal, ovarian, gastric), tracing tumor progression from precursor lesions to invasive NEC. Genomic analyses revealed a shared trunk of driver mutations across all tumor compartments, confirming their clonal origin, while also uncovering additional compartment-specific alterations. Spatial transcriptomics, together with gene set enrichment analysis (GSEA), revealed distinct transcriptional profiles aligned with histologically annotated compartments (e.g., adenocarcinoma, NEC, precursor). In NECs, GSEA consistently showed downregulation of immune-related pathways and upregulation of proliferation-associated pathways compared to non-neuroendocrine tumor areas. Moreover, distinct transcriptomic subclusters were identified within morphologically homogeneous NEC regions in two of the three cases. These subclusters exhibited significant differences in immune regulation, proliferation signaling, and cell-cycle control, and were associated with divergent predicted chemotherapy-response signatures, suggesting clinically relevant implications for treatment sensitivity and resistance. In summary, our findings indicate that despite a shared clonal origin, MiNEN develop distinct genetic and transcriptomic features across tumor compartments. The inconsistent presence of transcriptomic subclusters within morphologically similar regions underscores the complexity of intratumoral heterogeneity in these aggressive neoplasms. By connecting morphological and molecular layers of tumor architecture, spatial profiling may aid in translating biological complexity into more targeted clinical strategies.