Rapid, Sustained B Cell Receptor Signaling in Lymphoma B Cells Differs from Normal Signaling in Tumor Infiltrating Nonmalignant B Cells.

The B cell receptor (BCR) drives life and death signaling throughout B cell development, and dysregulation of BCR signaling might be expected to play a role in aberrant proliferation of lymphoma B cells. We have previously used flow cytometry based cell signaling profiles to identify patterns of altered signaling in acute myeloid leukemia that were informative of clinical outcome (Irish et al., Cell, 2004). Here we used a similar signaling profiles approach to compare BCR signaling in normal and lymphoma B cells. However, in addition to comparing follicular lymphoma (FL) B cells with peripheral blood B cells from normal donors, we also interrogated signaling within individual non-tumor B cells infiltrating FL tumor biopsies. By staining for CD20 and BCR light chain isotype (κ vs. λ), we could distinguish tumor and normal B cells within each patient biopsy. Following crosslinking of BCR heavy chains (shared by tumor and non-tumor B cells), we measured phosphorylation of Syk and Btk proteins, as markers of early BCR signaling activity, and Erk1/2 and p38, as markers of downstream BCR signaling effector activity. The BCR signaling network in FL tumor B cells was activated more rapidly than infiltrating non-tumor B cells, achieved greater levels of per-cell signaling, and sustained high levels of signaling over a period of hours. In lymphoma B cells, BCR-mediated Btk and Erk1/2 phosphorylation could reach the normal maximum in as little as 4 minutes, which was much more rapid than the 30–60 minutes required for peak signaling in non-tumor B cells. Strikingly, the timing and magnitude of BCR pathway protein phosphorylation we measured in non-tumor B cells within tumor biopsies was the same as that of normal, mature B cells from peripheral blood. These results suggest that the altered BCR signaling we identified in lymphoma is cell-intrinsic and associated with lymphomagenesis, as opposed to being a general change in tumor microenviornment affecting all B cells within a biopsy. FL tumor B cells from different patients were distinguished by the degree and number of changes to BCR signaling, such that variable profiles of lymphoma signaling kinetics distinguished each patient from the consistent signaling of normal B cells. These results identify cell-intrinsic changes to BCR signaling that may contribute to immortalization of lymphoma B cells and suggest that single cell profiles could identify lymphoma specific BCR-mediated signaling responsible for clinical outcomes.