Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma

Abstract

The two major subtypes of diffuse large B cell lymphoma (DLBCL)--activated B cell-like (ABC) and germinal center B cell-like (GCB)--arise by distinct mechanisms, with ABC selectively acquiring mutations that target the B cell receptor (BCR), fostering chronic active BCR signaling. The ABC subtype has a ∼40% cure rate with currently available therapies, which is worse than the rate for GCB DLBCL, and highlights the need for ABC subtype-specific treatment strategies. We hypothesized that ABC, but not GCB, DLBCL tumors would respond to ibrutinib, an inhibitor of BCR signaling. In a phase 1/2 clinical trial that involved 80 subjects with relapsed or refractory DLBCL, ibrutinib produced complete or partial responses in 37% (14/38) of those with ABC DLBCL, but in only 5% (1/20) of subjects with GCB DLBCL (P = 0.0106). ABC tumors with BCR mutations responded to ibrutinib frequently (5/9; 55.5%), especially those with concomitant myeloid differentiation primary response 88 (MYD88) mutations (4/5; 80%), a result that is consistent with in vitro cooperation between the BCR and MYD88 pathways. However, the highest number of responses occurred in ABC tumors that lacked BCR mutations (9/29; 31%), suggesting that oncogenic BCR signaling in ABC does not require BCR mutations and might be initiated by non-genetic mechanisms. These results support the selective development of ibrutinib for the treatment of ABC DLBCL.

Trial registration: ClinicalTrials.gov NCT00849654 NCT01325701.

Figures

Figure 1

Tumor response to ibrutinib therapy. (a) Waterfall plot of maximum change from baseline of SPD of lymph nodes for subjects with evaluable tumors (n = 43). (b) Overall response rate by DLBCL subtype. Fisher’s exact test of the overall response rate between the ABC and GCB groups (*P = 0.0106). Fractions above the bars represent the number of subjects showing a complete or partial response (numerator) over the total number of subjects in the group (denominator). (c) Kaplan–Meier analysis of progression-free survival. Log-rank test for analysis between groups (**P = 0.0038). (d) Kaplan–Meier analysis of overall survival. Log-rank test for analysis between groups (***P = 0.056). ABC, activated B cell–like; GCB, germinal center B cell–like; DLBCL, diffuse large B cell lymphoma; CR, complete response; OS, overall survival; PFS, progression-free survival; PR, partial response; SPD, sum of the product of the diameters.

Figure 2

Influence of B cell receptor mutations on ibrutinib response in ABC DLBCL. (a) Overall response rate according to CD79B mutational status. Fractions above the bars represent the number of subjects showing a response (complete or partial response; numerator) over the total number of subjects in the group (denominator). (b) PCR analysis of a region of CD79A with a 45-bp deletion in genomic DNA from tumors taken before ibrutinib therapy (pre-Rx) and again after 3 d of ibrutinib therapy (3 d on Rx). (c) Sanger sequencing analysis of tumor DNA as in b. The deletion of a splice acceptor site and part of the ITAM region is indicated. (d) Surface IgM expression in the indicated DLBCL lines in which endogenous CD79A expression was knocked down and cells were reconstituted with the indicated CD79A isoforms. Gating on a co-transduced Lyt2 marker identified the subset of transduced cells with equivalent ectopic CD79A RNA expression. (e) The OCI-Ly10 ABC DLBCL line was transduced with exogenous CD79A isoforms as in d and were treated with the indicated concentrations of ibrutinib for 4 d. Viability of cells relative to DMSO-treated cells is displayed as the mean from three biological repeats. Error bars denote s.e.m. Data from b and c are from single experiments, whereas data in d are representative of three biological repeats. DMSO, dimethyl sulfoxide; PCR, polymerase chain reaction.

Figure 3

Influence of recurrent genetic alterations on ibrutinib response in ABC DLBCL. (a) Overall response rates by MYD88 mutation status. Fisher’s exact test of the overall response rate between the MYD88 mutant and non-mutant group (P = 0.493). Fractions above the bars represent the number of subjects with a response (complete or partial response; numerator) over the total number of subjects in the group (denominator). (b) Overall response rates by CD79B and MYD88 mutation status. Fisher’s exact test of the overall response rate between the CD79B mutant/MYD88 mutant group and the CD79B wild type/MYD88 mutant group (*P = 0.01). (c) Toxicity of ibrutinib for cell-line models of ABC and GCB DLBCL harboring genetic lesions in CD79A, CD79B and MYD88, as indicated. Cells were treated for 3 d with ibrutinib at the indicated concentrations and assessed for viability as described. Error bars denote s.e.m. of triplicates. (d) MYD88 potentiates chronic active BCR signaling in ABC DLBCL. The indicated ABC DLBCL lines were treated with the MYD88 dimerization inhibitor IMG-2005 (100 μM) or a control peptide for 16 h and analyzed by immunoblot for the indicated proteins. Data are representative of three biological repeats. (e) MYD88 knockdown reduces chronic active BCR signaling in ABC DLBCL. TMD8 and HBL1 ABC DLBCL cells were transduced with the indicated shRNAs, induced to express the shRNAs with doxycycline for 48 h, and evaluated by immunoblot analysis for the indicated proteins. Data are representative of independent experiments in HBL1 (n = 7) and TMD8 (n = 10). (f) Overall response rates by CARD11 mutation status. Fractions above the bars represent the number of subjects with a response (complete or partial response; numerator) over the total number of subjects in the group (denominator). (g) Overall response rates by TNFAIP3 status. TNFAIP3 inactivation denotes TNFAIP3 nonsense or frameshift mutation, TNFAIP3 double deletion, or TNFAIP3 mRNA <2 s.d. below the mean of ABC DLBCL samples. TNFAIP3 WT denotes cases without these TNFAIP3 alterations. Fractions above the bars represent the number of subjects with a response (complete or partial response; numerator) over the total number of subjects in the group (denominator). (h) Theoretical model of ABC DLBCL pathogenesis indicating BCR-dependent and BCR-independent genetic pathways. Shown at the left are three genetic scenarios that can be associated with chronic active BCR signaling and ibrutinib sensitivity. Hypothetically, the BCRs in these tumors could be engaged by an antigen (turquoise hexagons) and hence are clustered. The right of this figure illustrates ibrutinib-resistant ABC DLBCL tumors that do not rely on chronic active BCR signaling, but rather use a mutant MYD88 isoform to engage the NF-κB pathway. The BCRs in these tumors are therefore depicted as unclustered and not engaged by an antigen. Pink asterisks (*) indicate activating mutations. ABC, activated B cell–like; BCR, B cell receptor; CR, complete response; DLBCL, diffuse large B cell lymphoma; PR, partial response.