We used clustered regularly interspaced short palindromic repeats/Cas9-mediated genomic modification to investigate B-cell receptor (BCR) signaling in cell lines of diffuse large B-cell lymphoma (DLBCL). Three manipulations that altered BCR genes without affecting surface BCR levels showed that BCR signaling differs between the germinal center B-cell (GCB) subtype, which is insensitive to Bruton tyrosine kinase inhibition by ibrutinib, and the activated B-cell (ABC) subtype. Replacing antigen-binding BCR regions had no effect on BCR signaling in GCB-DLBCL lines, reflecting this subtype's exclusive use of tonic BCR signaling. Conversely, Y188F mutation in the immunoreceptor tyrosine-based activation motif of CD79A inhibited tonic BCR signaling in GCB-DLBCL lines but did not affect their calcium flux after BCR cross-linking or the proliferation of otherwise-unmodified ABC-DLBCL lines. CD79A-GFP fusion showed BCR clustering or diffuse distribution, respectively, in lines of ABC and GCB subtypes. Tonic BCR signaling acts principally to activate AKT, and forced activation of AKT rescued GCB-DLBCL lines from knockout (KO) of the BCR or 2 mediators of tonic BCR signaling, SYK and CD19. The magnitude and importance of tonic BCR signaling to proliferation and size of GCB-DLBCL lines, shown by the effect of BCR KO, was highly variable; in contrast, pan-AKT KO was uniformly toxic. This discrepancy was explained by finding that BCR KO-induced changes in AKT activity (measured by gene expression, CXCR4 level, and a fluorescent reporter) correlated with changes in proliferation and with baseline BCR surface density. PTEN protein expression and BCR surface density may influence clinical response to therapeutic inhibition of tonic BCR signaling in DLBCL.

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Introduction. Targeting BCR signaling with the BTK inhibitor ibrutinib is clinically effective against most B-cell lymphomas, including the activated B-cell (ABC) subtype of diffuse large B-cell lymphoma (DLBCL), but not the germinal center B-cell (GCB) subtype. Active BCR signaling in GCB-DLBCL was suggested by studies with a Syk inhibitor and our previous studies using BCR knockout (KO). We addressed these questions: why is the BCR active in DLBCL, and how does it signal in GCB-DLBCL? Methods. We used CRISPR/Cas9 technology to modify selected genes by KO or homologous recombination-mediated knock-in (KI). For some genes KI was used to express a fluorescent protein (FP; e.g., GFP) instead of the targeted gene (KI/KO), or to modify the targeted gene together with KI of an FP, for detection of modified cells. Results. In GCB lines (OCI-Ly7 and OCI-Ly19) and ABC lines (U2932 and HBL-1), we simultaneously replaced the hypervariable region (HVR) exons of both immunoglobulin heavy (IgH) and light chains (IgL) with HVR sequences from normal B cells recognizing tetanus toxoid (TT). GFP and CFP respectively marked KI of IgH and IgL HVRs, and KI of the endogenous HVR sequences in each line served as controls. In CFP+/GFP+ cells, the TT specific BCR (TT-BCR) was expressed at similar or higher levels than the endogenous BCR (endo-BCR) and was functional, as shown by calcium flux in response to TT. The TT-BCR maintained growth of GCB lines (Fig. 1), indicating that they use "tonic", antigen-independent BCR signaling. Other features of tonic signaling were confirmed in more GCB lines: 1) the toxicity of BCR KO, which eliminates AKT S473 phosphorylation, was rescued by PTEN KO or expression of constitutively active AKT (mAKT), showing that BCR signaling serves principally to activate PI3K/AKT; and 2) KO of SYK or CD19, or truncation or ITAM mutation of the cytoplasmic tail of CD79A, none of which affect surface BCR levels, were as toxic as BCR KO but were non-toxic in BCR/PTEN double-KO cells. In contrast, the TT-BCR was as growth-slowing as BCR KO to the ABC line U2932 (Fig. 1), and substantially toxic to HBL-1, indicating that BCR signaling is self antigen-dependent in ABC-DLBCL. Reversion of somatic hypermutations in the U2932 HVRs was also as growth-slowing as BCR KO (Fig. 1), suggesting that self-antigen reactivity developed during BCR affinity maturation. Tonic signaling by the TT-BCR provided a detectable benefit (as compared to BCR KO) in PTEN-expressing HBL-1, whereas there was no difference between TT-HVR BCR and BCR KO in PTEN-deficient U2932. The surface TT-BCR level was higher than the endo-BCR level in ABC lines, and dropped with TT stimulation, suggesting that endo-BCRs in ABC lines undergo constant antigen stimulation with BCR internalization. The presumed self-antigen in ABC lines seems to be cell line-specific, since HVRs from ABC lines TMD8 and HBL-1 did not rescue growth of U2932. BCR KO in ABC lines was also not rescued by PTEN KO or mAKT. In cells whose BCRs were labeled by KI to fuse GFP to CD79A, super-resolution microscopy showed macro-clustering of BCR complexes at the surface of ABC line HBL-1, not seen in GCB lines (Fig. 2). Several findings suggested the clinical potential of targeting tonic BCR signaling in DLBCL: 1) clinical trial-stage inhibitors of SYK (P505-15) and PI3K (idelalisib) were toxic to GCB lines (less so with PTEN KO); 2) GCB lines (6/8) were sensitized by BCR KO to an in vitro CHOP-like regimen; 3) P505-15 or idelalisib sensitized GCB lines (3/3) to CHOP in vitro; and 4) evidence of tonic signaling in ABC line HBL-1 after removing antigen-driven signaling by HVR replacement. Conclusion. The BCR provides antigen-independent tonic signals to activate PI3K/AKT in GCB-DLBCL and antigen-dependent signaling in ABC-DLBCL. Targeting of B-cell specific tonic signling alone or in combination could be clinically effective in both types of DLBCL. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 1. Effect of BCR KO or HVR replacement in OCI-LY19 (A) and U2932 (B) cell lines. Endogenous IgH and IgL HVRs were replaced with HVR pairs (TT3 and/or TT6) recognizing tetanus toxoid, reverted to undo the effect of SHM, or restored with original HVRs. Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Figure 2. Representative super-resolution images of BCR localization in live DLBCL cells. BCR labeled by CD79A-GFP fusion, surface membrane by CellMask staining. (bars = 5 µ m) Disclosures Westin: Spectrum: Research Funding.

Introduction The causal association between H. pylori and gastric MALT lymphoma has been well demonstrated and H. pylori eradication with antibiotics has emerged as the standard therapy for Stage I H. pylori positive (HPP) gastric MALT lymphoma. As per the NCCN Guidelines, radiation therapy is the preferred treatment option for early stage H. pylori negative (HPN) gastric MALT lymphoma and antibiotic therapy is not indicated. However, successful treatment of HPN early stage gastric MALT lymphoma with antibiotics was reported in small series of patients by Raderer et al, Gut, 2006 [(5/6 patients achieved complete remission (CR)] and Asano et al, Tohoku J Exp Med, 2012 (5/17 patients achieved CR). Here, we report the outcome of Stage I HPP and HPN gastric MALT lymphoma patients treated with antibiotics at MD Anderson Cancer Center, Houston, Texas, USA over a period of 20 years. Methods We reviewed medical records of all pathologically proven gastric MALT lymphoma patients (n=128) referred to MD Anderson Cancer Center at initial diagnosis between 1991 and 2011. Only patients with Stage IAE disease were considered for this analysis. Patients with large cell transformation were excluded. H. pylori status was determined by histopathology and serum antibody assay. Clinical staging was determined by upper GI endoscopy with biopsy, bone marrow biopsy, and CT scans of neck, chest, abdomen, and pelvis and/or PET-CT scan. Response was assessed by upper GI endoscopy every 3-6 months until complete resolution of lymphoma. Complete remission was defined as the absence of histopathologic evidence of lymphoma on endoscopic biopsies. Results Of the 128 patients reviewed, 81 patients had Stage IAE disease without histologic evidence of large cell transformation. The 81 Stage IAE patients were assigned to HPP (39/81, 48%) or HPN group (42/81, 52%) based on histopathologic evidence of H. pylori. The higher-than-expected proportion of HPN patients might be due to referral bias to a tertiary care cancer center. The results of H. pylori antibody serology are shown in Table 1. There was no significant difference in age, gender, and race between HPP and HPN groups. First-line antibiotic therapy was administered for all 39 (100%) HPP patients and 28/42 (67%) HPN patients. The CR rate after antibiotic therapy was significantly higher in HPP compared with HPN patients (22/39, 56% vs 7/28, 25%; p=0.019). The median time to achieve a CR was similar for the two groups (HPP: 7.8 mo, range 3-40 mo; HPN: 9.7 mo, range 3-35 mo; p=0.385). After a median follow-up of 110 mo for the HPP group and 91.5 mo for the HPN group, 3/22 (14%) and 2/7 (28%) responders relapsed, respectively (p=0.362). Patients who failed to achieve a CR with antibiotic therapy were mostly treated with radiation: 14/17 in the HPP group and 19/21 in the HPN group. All patients that received radiation achieved a CR. Of the patients in both groups who received upfront antibiotic therapy, there were no differences in time to progression (HPP, 90% vs HPN, 92% at 8 years; p=0.543) and overall survival (HPP, 93% vs HPN, 100% at 8 years; p=0.068). Conclusions Our results demonstrate that a substantial proportion of patients with Stage IAE HPN gastric MALT lymphoma achieve durable remission with antibiotic therapy alone. It is possible that such responses may be because of false-negative H. pylori test results or due to association of HPN gastric MALT lymphoma with other unidentified bacteria. We also observed that HPN gastric MALT lymphoma patients failing antibiotic therapy could be effectively salvaged with radiation therapy and their long-term outcome is not affected by delay from initial trial of antibiotic therapy. Thus, the results of this largest series-to-date of HPN patients treated with first-line antibiotic therapy, combined with results of smaller series reported previously, suggest that 1) antibiotic therapy should be considered as first-line therapy for Stage IAE HPN gastric MALT lymphoma patients, and 2) radiation therapy could be avoided in a subset of these patients. Table 1. H. pylori status in Stage IAE gastric MALT lymphoma as determined by histopathology and H. pylori antibody. H. pylori positive by histology (n=39) H. pylori negative by histology (n=28) CR (n=22) Non-CR(n=17) CR (n=7) Non-CR(n=21) H. pylori Ab Positive 4 4 0 3 H. pylori Ab Negative 8 2 1 7 Not available 10 11 6 11 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.