Signaling Diversity in Human Lymphoma B Cells and in Tumor Infiltrating T Cells Correlates with Follicular Lymphoma Patient Clinical Outcomes

Introduction: Flow cytometry analysis of live cells from cryopreserved tumor samples provides the opportunity to study simultaneously the biology of both cancer cells and the patient’s immune system cells. Furthermore, single cell based approaches can identify and quantify subsets within mixed populations, such as heterogeneous primary tumors, without the need to physically separate cells. We have previously used flow cytometry to identify differences in signaling mechanism between healthy human B cells from two differentiation stages and to compare signaling kinetics of tumor and non-malignant B cells within primary human lymphoma specimens¹.

Methods: Here we used barcoded phospho-specific flow cytometry to measure signaling events in live lymphoma B cells and tumor-infiltrating T cells from follicular lymphoma (FL) tumor samples obtained before patients received therapy. Patients were then stratified according to signaling and clinical outcome was examined within the resulting groups. We examined response to initial chemotherapy, which was a combination of cyclophosphamide, vincristine, and prednisone (cvp), and overall survival, measured as the time from diagnosis to last follow up or mortality.

Results: Differences in B cell receptor (BCR) signaling strength and kinetics characterized previously unappreciated diversity within the lymphoma B cell population. In some cases, BCR crosslinking triggered robust phosphorylation of AKT and ERK in one subset of lymphoma cells, while in another lymphoma subset within the same tumor sample, BCR crosslinking did not lead to phosphorylation of either protein. In these cases, both lymphoma B cell subsets expressed BCL2 and surface immunoglobulin heavy and light chain restricted to the tumor isotype. Patients whose lymphoma tumor contained a BCR insensitive cell subset (Group 2) had significantly worse responses to cvp therapy (p = 0.001) and lower overall survival (p = 0.003) than patients whose lymphoma cells displayed more homogeneous BCR signaling (Group 1). We next examined tumor infiltrating T cell signaling in the FL cases from Group 1, where no significant BCR insensitive subset was observed. Within Group 1, differences in the magnitude of IL-2, IL-7, and IL-15 mediated STAT5 phosphorylation in tumor infiltrating T cells further distinguished a set of patients with significantly higher overall survival (p = 0.04).

Conclusions: These results identify BCR signaling in lymphoma B cells and cytokine signaling in tumor infiltrating T cells as clinically relevant biomarkers for tracking and isolating lymphoma cell subsets and for monitoring immune system activity during therapy. By following patients over time, we can now determine whether cell intrinsic signaling diversity enables the emergence of therapy insensitive cancer cell subsets.