NF-κB Signaling In Response to CpG Stratifies Mantle Cell Lymphoma Patient Outcome

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin's lymphoma. Although newer treatment regimens including intensive chemo-immunotherapy followed by stem cell transplant have improved survival to a median approaching 6 years, MCL is still incurable in many cases. The use of flow cytometry to investigate stimulation induced signaling at the single cell level represents an opportunity to obtain each patient's signaling profile as well as to discover tumor cell heterogeneity and identify signaling events potentially responsible for poor outcomes. We recently used this method to characterize signaling in subpopulations of tumor samples from patients with follicular lymphoma (FL). In FL, we identified a lymphoma negative prognostic (LNP) cell subset with impaired B cell antigen receptor (BCR) signaling, the prevalence of which correlated with adverse clinical outcome (Irish et al., PNAS 2010). In the present study we used the same approach to identify signaling responses with large variation within MCL patient samples, and tested whether these signaling events might predict patients' clinical outcomes. By understanding the biology based on lymphoma signaling profile, we might identify prognostic significant factors such as the LNP subset observed in FL.

Single cell flow cytometry measurements of signaling were acquired for samples of MCL (n=25). Of these, 21 MCL specimens were obtained prior to any treatment. Median age was 61 years, and follow up time ranged from ½ until 15 years. Treatment regimens varied. Samples from diffuse large B cell lymphoma (DLBCL, n=12), FL (n=14), chronic lymphocytic leukemia (CLL, n=14) and tonsils from healthy donors (n=4) were also included for comparison. Phosphorylation of 14 signaling proteins was measured under 12 different stimulation conditions in every cell within the patient specimens, including lymphoma B cells and in tumor-infiltrating T cells. Stimulation conditions included BCR crosslinking, CD40 ligand, CpG oligodeoxynucleotides (CpG ODN) and several cytokines (IL-4, IL-10, IL-21, IFN-γ).

The magnitude of most cytokine induced signaling responses in lymphoma cells from MCL patients was higher than in lymphoma cells from FL. This included IL-10 induced phosphorylation of STAT3 (p<0.0001) and IL-4 induced phosphorylation of STAT6 (p<0.0001). In contrast, IL-21 induced phosphorylation of STAT3 in lymphoma cells was similar in FL and MCL patients. Furthermore, MCL tumor cells responded better to BCR crosslinking at 9 measured downstream signaling proteins, including SYK, Src family kinases (SFK) and PLC-γ, than tumor cells from FL and CLL patients. Although signaling responses overall were higher in MCL, there was a large variation in the lymphoma cells' signaling capacity within each lymphoma histology. To identify possible signaling events that correlated with clinical outcome in MCL, we first identified the signaling events with the largest variance. This included phosphorylation of SYK and PLC-γ by BCR crosslinking, phosphorylation of NF-κB (p65) induced by CpG ODN, CD40L, or by PMA and ionomycin, and phosphorylation of STAT3 induced by IL-10 and IL-21. Of these seven signaling events, only CpG ODN induced p-p65 significantly stratified overall survival when MCL patients were split into two even groups based on higher and lower signaling response, compared to healthy B cells from tonsils. Hence, patients whose MCL cells displayed high CpG ODN induced NF-κB (p65) signaling had an adverse prognosis (p<0.008, HR=5.1). In contrast with our observations of FL, BCR signaling did not stratify the outcome of MCL patients.

Potentiated NF-kB p65 signaling in response to CpG ODN was associated with poor prognosis in MCL. The tumor cells capacity to respond to CpG induced signaling likely represents a biological advantage for the tumor cells in this lymphoma entity.

No relevant conflicts of interest to declare.