Cytokine Profiling of ABC-Subtype and GCB-Subtype Diffuse Large B Cell Lymphoma: Systemic Nfkb Activation and Impact on Myeloid-Derived Suppressor Cells Distribution

Background. Among patients with newly diagnosed diffuse large B cell lymphoma (DLBCL), undergoing standard immunochemotherapy R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone), overall survival is significantly worse for those with activated B cell-like (ABC) subtype compared with germinal center B cell-like (GCB) subtype. Among the key differences between DLBCL subtypes, constitutive activation of nuclear factor kappa B (NFκB) in the ABC-subtype has been associated with a rise of circulating myeloid-derived suppressor cells (MDSC).

Patients and Methods. In this pilot study, blood samples were collected from 6 ABC-subtype and 5 GCB-subtype newly diagnosed DLBCL patients prior to and after R-CHOP therapy. To assess the systemic impact of constitutive NFκB activation in ABC-subtype compared with GCB-subtype DLBCL, serum concentrations of 27 immune analytes (15 NF-κB-dependent chemokines, cytokines and growth factors, 12 NFκB-independent chemokines and cytokines) were measured retrospectively using a BioPlex platform. ABC-subtype and GCB-subtype DLBCL immune profiles were further investigated using a 6-color flow cytometry panel (BD FACS Aria II), in which peripheral blood samples were prospectively analyzed for expression of CD11b, HLA-DR, CD33 and IL-4R to define MDSC (CD11b⁺, HLA-DR^-/low, CD33⁺, IL-4R⁺), either monocytic (IL-14⁺) or granulocytic (CD15⁺).

Results. Among the 11 patients enrolled in the study, 7 (5 ABC and 2 GCB) have completed R-CHOP therapy and are in complete remission. Twenty out of 27 immune analytes surveyed to establish DLBCL immune profiles were either not detected or constitutively expressed among ABC and GCB patients (pre or post therapy). At baseline, the predominant difference between DLBCL subtypes was centered around 3 NFκB-independent immune analytes. The IRF4-dependent chemokine CXCL10 was highly expressed in ABC-subtype DLBCL while elevated IL-12p70 and IL-13, (T-bet and Gata3-dependent cytokines, respectively) were mainly found in GCB-subtype DLBCL. Post R-CHOP therapy, serum concentrations of NFκB-dependent innate immune mediators, the cytokine IL-6 and the chemokine CXCL8, decreased by »50% regardless of DLBCL subtypes. While no systemic difference in NFκB-dependent immune analytes could distinguish the ABC-subtype from GCB-subtype DLBCL, circulating gMDSC and mMDSC were 8 and 3 times more prevalent among ABC patients. Following R-CHOP, their levels decreased to those observed in patients with the GCB subtype.

Conclusions. Although constitutive NFκB activation is the hallmark of ABC-subtype DLBCL, no systemic NFκB-dependent immune analyte could distinguish ABC from GCB-subtype DLBCL in this pilot study. Despite the lack of a "NFκB immune signature", MDCSs (especially mMDSC) were predominantly found in ABC-subtype DLBCL. Significant differences were also observed among NFκB-independent immune analytes, predominantly adaptive cytokines, indicating GCB-subtype DLBCL have a more profound effect on T cell polarization, specifically TH2, than ABC-subtype.