A phospho-specific flow cytometry study reveals the impact of BCR signaling activation in mantle cell lymphoma progression and therapy

Mantle cell lymphoma (MCL) is a rare and heterogeneous subtype of Non-Hodgkin lymphoma characterized by the expansion of mature B-cells in the mantle zone of the lymph nodes. Recent evidence supports that B-cell receptor (BCR) signaling is pivotal for MCL initiation and progression and is a target for therapeutic treatment. However, drug resistance inevitably emerges. In this study, we characterized the activation status of phosphoproteins on the BCR pathway, and evaluated the impact of the BCR in MCL progression and response to therapy. We measured the activation status of 9 BCR signaling kinases (SYK, LCK, BTK, PLCγ2, p38, ERK1/2, AKT, NF-κB p65, STAT5) in peripheral blood mononuclear cells (PBMC) from 30 MCL patients and 10 healthy donors, in the basal and anti-IgM modulated conditions, at single cell level using phospho-specific flow cytometry. BCR modulation with anti-IgM determined a heterogeneous activation of the BCR signaling among patients’ samples, with identification of two clusters showing differential responses to BCR stimulation. The cluster comprising samples with higher BCR signaling response (HR) was associated with shorter survival than samples grouped in the lower BCR signaling response (LR) cluster. This finding was further corroborated with time-to-event analyses, which showed that the MCL international prognostic index (MIPI) high-risk category together with high STAT5 response to the stimulation were significant predictors of shorter PFS and OS. In addition, MIPI high-risk category combined with high SYK response predicted shorter OS. While basal BCR activation did not provide prognostic information, higher constitutive activation of AKT was predictive of inferior response to the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib. In conclusion, we identified BCR signaling activation profiles that were associated with poorer clinical outcome and resistance to ibrutinib. This study highlighted the prognostic and predictive significance of BCR activity in MCL and advanced our understanding of signaling heterogeneity underlying clinical behavior of MCL. A future challenge is the integration of BCR signaling data with genetic signature to predict patient’s clinical behavior and drug response for a more personalized treatment approach. We thank Fondazione Italiana Linfomi (PGR Ed. 2019) for funding support.