DP048 | PLASMA EXTRACELLULAR VESICLES CARRY A CIRCULATING DISEASE-SPECIFIC SIGNATURE OF HAIRY CELL LEUKEMIA

Introduction: Classic Hairy Cell Leukemia (HCL) is a rare and incurable lymphoproliferative B-cell disorder characterized by infiltration of hairy cells within bone marrow, liver and spleen. Clinical features are frequent relapses and potentially life-threatening infections. No disease-specific biomarkers with prognostic value have yet penetrated clinical practice. Small (S-; 50-200 nm) and Large (L-; 0.2-10 µm) extracellular vesicles (EVs) are particles promoting intercellular communication with a pivotal role in “education” of tumor niche. However, their role in HCL is unknown. Methods: S- and L-EVs were isolated from plasma of 7 HCL patients at diagnosis and 10 healthy donors (HD), and characterized for surface protein composition (flow cytometry) and mRNA cargo (RNA sequencing). Results: Flow cytometry analysis showed that, compared to HD EVs, HCL S- and L-EVs overexpress HCL markers CD19, CD20, CD25, CD11c, and adhesion-related molecule CD44. Moreover, HCL S-EVs were enriched in monocyte (CD14) and endothelial (CD105) markers. Exploring EV mRNA cargo, a clear separation was found between S- and L-EVs and between HCL and HD samples, supporting a selective mRNA packaging within HCL and HD EVs. Moreover, after splitting the genes by length, a strong difference was assessed between S- and L-EVs of both HCL and HD: S-EVs express all transcripts, while L-EVs rarely express the short ones. Gene Ontology (GO) analysis showed that comparing HCL and HD L-EVs, the most enriched terms were related to the immune B cell origin of the disease, like B cell receptor signaling pathway and lymphocyte differentiation, while comparing HCL and HD S-EVs, differentially expressed genes were related to cytoplasmic components and RNA splicing. Analysis of mRNA data also revealed significant differences in alternative splicing patterns between HCL and HD, with dominant transcript changes in approximately half of differentially spliced genes. In particular, GO analysis indicated that dysregulated splicing events were associated with intracellular signaling and transcription elongation-regulatory genes when comparing HCL and HD L-EVs, and post-transcriptional regulation and protein degradation genes when comparing HCL and HD S-EVs. Conclusions: Here we identified a tumor-associated signature of HCL EVs that provides pathogenesis-related information and highlights the diagnostic/prognostic potential of EV biomarkers for future liquid biopsy approaches in HCL and other rare tumors.