CO10 | Spatial transcriptomic analysis of chronic lymphocytic leukemia lymph nodes reveals tumor microenvironment evolution during disease progression

Introduction. In chronic lymphocytic leukemia (CLL), malignant cells bidirectionally interact with the tumor microenvironment (TME), creating pro-tumoral niches within lymph nodes (LNs) that sustain disease progression and therapy resistance. Understanding dynamic CLL–TME modifications is crucial to identify novel biomarkers and actionable cell-to-cell interactions. We applied Digital Spatial Profiling (DSP) to characterize tumor and immune populations—including T lymphocytes and macrophages—while distinguishing microanatomical areas within proliferation centers (PCs) from surrounding non-PC regions.Methods. Seventeen FFPE CLL-infiltrated LNs were analyzed and grouped into three clinical subsets: A (n=6) indolent CLL; B (n=4) CLL requiring treatment within 6 months after LN biopsy; and C (n=7) relapsed/progressive CLL. Regions of interest (ROIs) were defined by multiplexed immunofluorescence using CD3, CD68, and MUM1 to identify T cells, macrophages, and neoplastic B cells, both inside and outside PCs. Normalized transcriptomic matrices were generated for each cell type and analyzed by Gene Ontology (GO) on differentially expressed genes (p0.2), ranking pathways by Normalized Enrichment Score (NES) with FDR