NF-κb / STAT3 Signaling Pathway Collaboration in Diffuse Large B-Cell Lymphoma (DLBCL) Cell-of-Origin (COO) Subtypes: Resolution of Myeloid Subversion By Standard Induction Chemotherapy

Background. Germinal center B-cell (GCB) and activated B-cell (ABC) are two main cell-of-origin (COO) subtypes of diffuse large B-cell lymphomas(DLBCL). Patients presenting with the ABC-DLBCL subtype have inferior outcomes following standard rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) therapy. Since ABC-DLBCL phenotype is characterized by marked activation of nuclear factor kappa B (NF-κB) signaling pathway, we hypothesized that inferior outcomes of patients with this COO subgroup treated with standard therapy were due to unresolved innate immune bias or myeloid subversion.

Methods. Immunological profiles were established for 23 newly diagnosed DLBCL patients (n=13 ABC, n=10 GCB) prior to induction therapy (R-CHOP or R-EPOCH). Serum concentrations of 25 cytokines, chemokines and growth factors, were quantified by a multiplex protein assay. Peripheral distribution of myeloid cells (CD11b⁺ CD33⁺), myeloid maturation (HLA-DR^+/-IL-4R^+/-) and myeloid subversion [monocytic (m) and granulocytic (g) myeloid-derived suppressor cells (MDSCs)] were established by flow cytometry. These 31 immune variables were reassessed after completion of induction therapy for 16 DLBCL patients (n=10 ABC, n=6 GCB). Linear regression was used to determine the association between each tested variable and COO or modified international prognosis index (R-IPI). Unsupervised hierarchical cluster analysis was then applied to diagnosis samples with the selected variables that were differentially expressed between ABC- and GCB-DLBCL subtypes (p<0.1) using complete linkage and Euclidean distances. Paired t-test was used to compare each immune variable before and after chemotherapy.

Results. At diagnosis, 14 of 25 immune analytes transcriptionally regulated by NF-κB were equally expressed between the two COO subgroups. ABC-DLBCL has lower PDGF-bb serum concentrations (p=.008). Two immune analytes regulated by the STAT3 signaling pathway, INFg-induced protein 10 (IP10) and IL-9 are differentially expressed among the two COO. While STAT3 positively regulates IP10, leading to elevated IP10 serum concentrations in ABC-DLBCL patients (p=0.003), it negatively regulates IL-9 leading to reduced IL-9 serum concentrations in this COO-subtype (p=0.05). Hierarchical clustering analysis of DLBCL patients at diagnosis shows that 9/13 ABC-DLBCL patients exhibit this STAT3 signature. STAT3 signaling pathway activation is also associated with myeloid immune subversion promoting gMDSC expansion in the ABC-DLBCL subtype (p=0.05). No correlations could be established between immunological bias at diagnosis in the 2 COO subtypes and R-IPI. Among the 16 patients who have completed chemotherapy (n=12 R-CHOP & n=4 R-EPOCH), none were refractory to induction therapy. ABC-DLBCL subtypes displayed the most profound immunological alterations compared with GCB-DLBCL, with decrease in serum concentrations of 7 immune analytes regulated by NF-κB (IL-6, IL-8, MIP1a, MIP1b, IL-1ra, GM-CSF and PDGF-bb). Standard chemotherapy leads to resolution of myeloid subversion and STAT3 bias as exemplified by normalization of the distribution of gMDSCs and IP10 and IL-9 serum concentrations between the two COO subgroups.

Conclusion. Immune profiling of newly diagnosed DLBCL patients identified distinct biological signatures associated with COO subtypes. Highly enriched NF-κB signaling activation in ABC-DLBCL patients was associated with a STAT3-driven cytokine signature (NF-κB/STAT3 collaboration) and myeloid subversion. Standard induction chemotherapy normalized immunological bias across COO DLBCL subgroups. Longer follow up with clinical correlations are in progress to determine the significance of myeloid subversion and the NF-kB/STAT3 signature at diagnosis in DLBCL.