Figure 1.
Activation of RelB is frequent in patients with de novo DLBCL irrespectively of their ABC or GCB subtype and is associated with worse overall survival. (A) Whole cell extracts from frozen samples of 66 cases of de novo DLBCL (from the LYSA LNH03 trial) were analyzed by using EMSA for alternative RelB NF-κB subunit DNA-binding activity. Four representative RelB- positive cases (2 ABC and 2 GCB) and one RelB-negative case are presented. For supershifts, extracts were incubated with either anti-RelB or anti-RelA antibodies before incubation with the labeled probe. RelA- and RelB-containing complexes are indicated. (B) Distribution of RelB DNA- binding activity as defined by EMSA among the GCB and ABC patients with DLBCL (n = 66). (C) Top: prevalence of RelB activation as defined by EMSA in ABC and GCB DLBCLs (n = 66). Bottom: prevalence of ABC and GCB DLBCLs within the positive RelB-binding activity subgroup. (D) Top: prevalence of dominant activation of the 2 canonical NF-κB subunits RelA and cRel as defined by EMSA in ABC and GCB DLBCLs (n = 66). Bottom: prevalence of ABC and GCB DLBCLs within the 2 dominant canonical NF-κB subunit RelA and cRel activation subgroups. (E) Subunit composition of the RelA- and RelB-containing complexes as determined by supershift analysis with the indicated antibodies. (F) Immunohistologic staining of DLBCL biopsy specimens with anti–NF-κB2/p52 and anti–NF-κB1/p50 antibodies, n = 54 of the 66 EMSA cases. A representative image showing nuclear localization of NF-κB2/p52 (top left) and NF-κB1/p50 (top right) is presented. Magnification, ×200. The cutoff used to score cases as nuclear positive for p52 and p50 was ≥30%. (G) Correlation analysis of RelB DNA-binding activity with nuclear p52 and p50 as evaluated in panel F; n = 54 patients. P value by Fisher’s exact test. Color codes indicate the presence or absence of the corresponding feature. (H) Kaplan-Meier plot of overall survival according to RelB DNA-binding status (LYSA LNH03 trial, EMSA cases, n = 66). P value by log-rank (Mantel-Cox) test. IHC, immunohistochemistry.

Activation of RelB is frequent in patients with de novo DLBCL irrespectively of their ABC or GCB subtype and is associated with worse overall survival. (A) Whole cell extracts from frozen samples of 66 cases of de novo DLBCL (from the LYSA LNH03 trial) were analyzed by using EMSA for alternative RelB NF-κB subunit DNA-binding activity. Four representative RelB- positive cases (2 ABC and 2 GCB) and one RelB-negative case are presented. For supershifts, extracts were incubated with either anti-RelB or anti-RelA antibodies before incubation with the labeled probe. RelA- and RelB-containing complexes are indicated. (B) Distribution of RelB DNA- binding activity as defined by EMSA among the GCB and ABC patients with DLBCL (n = 66). (C) Top: prevalence of RelB activation as defined by EMSA in ABC and GCB DLBCLs (n = 66). Bottom: prevalence of ABC and GCB DLBCLs within the positive RelB-binding activity subgroup. (D) Top: prevalence of dominant activation of the 2 canonical NF-κB subunits RelA and cRel as defined by EMSA in ABC and GCB DLBCLs (n = 66). Bottom: prevalence of ABC and GCB DLBCLs within the 2 dominant canonical NF-κB subunit RelA and cRel activation subgroups. (E) Subunit composition of the RelA- and RelB-containing complexes as determined by supershift analysis with the indicated antibodies. (F) Immunohistologic staining of DLBCL biopsy specimens with anti–NF-κB2/p52 and anti–NF-κB1/p50 antibodies, n = 54 of the 66 EMSA cases. A representative image showing nuclear localization of NF-κB2/p52 (top left) and NF-κB1/p50 (top right) is presented. Magnification, ×200. The cutoff used to score cases as nuclear positive for p52 and p50 was ≥30%. (G) Correlation analysis of RelB DNA-binding activity with nuclear p52 and p50 as evaluated in panel F; n = 54 patients. P value by Fisher’s exact test. Color codes indicate the presence or absence of the corresponding feature. (H) Kaplan-Meier plot of overall survival according to RelB DNA-binding status (LYSA LNH03 trial, EMSA cases, n = 66). P value by log-rank (Mantel-Cox) test. IHC, immunohistochemistry.

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