Transcriptomic remodeling of bone marrow mesenchymal stromal cells in pediatric B-cell acute lymphoblastic leukemia: a four-gene signature

Background: Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is molecularly heterogeneous and influenced by the bone marrow microenvironment. Mesenchymal stromal cells (MSCs) provide critical niche signals, yet their transcriptomic remodeling in B-ALL is poorly defined. This study aims to define a compact gene signature that complements blast-centric diagnostics. Methods: healthy), Random Forest feature ranking, consensus clustering, and Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) analyses were performed. Within-cohort discrimination used 5-fold cross-validated logistic regression with bootstrapped area under the receiver operating characteristic curve (AUC) confidence intervals (CIs) (CCN4 mapped to WISP1 on GPL570). Public single-cell RNA sequencing (scRNA-seq) data provided cellular context. Peripheral blood real-time quantitative PCR (RT-qPCR) served as an exploratory assessment of systemic expression, not MSC validation. Results: We identified 78 differentially expressed genes (DEGs) in B-ALL-associated MSCs. A four-gene MSC signature [Dickkopf WNT Signaling Pathway Inhibitor 1 (DKK1), Regulator of G Protein Signaling 2 (RGS2), Cellular Communication Network Factor 4/WNT1-Inducible Signaling Pathway Protein 1 (CCN4/WISP1), Lysozyme (LYZ)] distinguished active B-ALL MSCs from healthy MSCs with cross-validated AUC 0.883 (95% CI: 0.783-0.966) and separated samples into two transcriptional states with distinct pathway enrichment, including extracellular matrix and immune-related programs. Single-cell data localized RGS2 and LYZ to immune compartments, whereas DKK1 and CCN4/WISP1 were low, supporting stromal specificity. Peripheral blood RT-qPCR showed decreased RGS2 and LYZ, whereas CCN4 showed a nonsignificant upward trend (P=0.053), underscoring compartment-dependent patterns. Conclusions: These data delineate transcriptional remodeling of bone marrow MSCs in pediatric B-ALL and nominate a four-gene MSC-associated signature. By centering on stromal MSC transcriptomes rather than leukemic blasts, we target a complementary microenvironmental layer of B-ALL biology that is underrepresented in blast-centric transcriptomics. The findings are hypothesis-generating and require independent MSC cohorts and functional validation.