B-CELL RECEPTOR SIGNALING PROFILES IDENTIFY SUBSETS OF MANTLE CELL LYMPHOMA PATIENTS WITH DIFFERENT CLINICAL OUTCOMES

Introduction. B cell receptor (BCR) signaling is a driving event in the development and progression of Mantle Cell Lymphoma (MCL). Recent evidence supports that BCR signaling is involved in drug resistance mechanisms and is a target for therapy. As clinical features and responses vary across patients, the aim of our research is to explore the variability of BCR signaling in MCL and unveil possible relationship with clinical outcome. Methods. We measured the phosphorylation status of nine BCR signaling phosphoproteins, namely pSYK, pLCK, pBTK, pPLCγ2, pp38, pERK1/2, pAKT, pNF-κB p65, pSTAT5, at the single cell level using phospho-specific flow cytometry combined with fluorescent cell barcoding (FCB) in peripheral blood mononuclear cells (PBMCs) from 10 MCL patients collected at diagnosis. The phosphorylation status of signaling proteins was measured in the basal condition and following BCR stimulation with anti- IgG, anti-IgD, anti-IgM antibodies, or their combination. Fluorescence intensity values were expressed as inverse hyperbolic sine (arcsinh) and corrected for the autofluorescence signals. Responses to BCR stimulation were calculated relative to signals in the basal condition. Then, arcsinh transformed data were subjected to unsupervised hierarchical cluster analysis (HCA) within the MCL samples. Association between signaling-defined groups of patients and clinical parameters was performed using Fisher’s exact test. Results. Analysis of signaling phosphoproteins in the basal condition revealed high levels of pBTK, pPLCγ2, and pSTAT5, with no or very low basal levels of pSYK, pLCK, pAKT and pNF-κB p65. After BCR stimulation with anti-IgM, we showed an overall significant higher level of both BCR proximal (pSYK, pLCK, pBTK, pPLCγ2) and more distal (pp38, pERK1/2, pAKT) signaling nodes compared to unstimulated, excepted for pNF-κB p65 and pSTAT5. In contrast, anti-IgG or anti-IgD stimulation did not induce phosphoproteins activation, except for pAKT, which was significantly activated after anti-IgG stimulation. Unsupervised HCA identified two main clusters of MCL patients: cluster 1 comprised samples with a lower signaling response, whereas cluster 2 included samples showing a higher signaling response. Remarkably, the BCR signaling-based clustering was significantly associated with clinical https://sies.abstracts.it/Autori/DettagliAbstract.php?RecID=G+gYzEURYh2mpguStNGMUQ== Pagina 1 di 2 30/11/21, 19:04 parameters including age, response to therapy, and progression of disease (POD) (Figure 1). Conclusions. Our results suggest that individual differences in BCR-signaling profiles can reflect clinical outcomes and responses to therapy, including BCR-signaling inhibitors, thus highlighting the predictive significance of BCR-signaling profiles in MCL. We thank Fondazione Italiana Linfomi (PGR Ed. 2019) and Centro Piattaforme Tecnologiche (CPT) of Verona University for technical support.