Germinal Center B-cell Diffuse Large B-cell Lymphoma Research Articles

Abstract B079: Molecular determinants of sensitivity to Polatuzumab-Vedotin

Abstract Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a sialylated glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V. Citation Format: Sean R Corcoran, Jaewoo Choi, Rachel E Fenner, Xin Yu, Sebastian Scheich, Galina Schevchenko, Evangelia K Papachristou, Vivian M Morris, Kamal Kishore, Clive S D'Santos, Stefania Pittaluga, George Wright, Jagan Muppidi, Daniel J Hodson, Louis M Staudt. Molecular determinants of sensitivity to Polatuzumab-Vedotin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B079.

Molecular Determinants of Sensitivity to Polatuzumab-Vedotin in Diffuse Large B-Cell Lymphoma

Purpose: Polatuzumab Vedotin (Pola-V) is an antibody-drug conjugate directed to the B cell surface antigen CD79B. When combined with conventional immunochemotherapy, Pola-V improves outcomes in DLBCL overall; however, there is noted heterogeneity in response to Pola-V, with germinal center b-cell (GCB) DLBCL showing no added benefit to the addition of Pola-V compared to standard immunochemotherapy. We aimed to identify molecular determinants of sensitivity or resistance to Pola-V. We hypothesized that these might lead us to innovative strategies to improve on-target tumor killing by Pola-V, or to find novel biomarkers that predict drug resistance. Methods: We employed combined drug-sensitization and CD79B-sorted CRISPR-Cas9 screening to identify molecular determinants of sensitivity to CD79B-directed, tumor killing by Pola-V in 9 cell lines representing different molecular subtypes of DLBCL. Results: Our results reveal the striking impact of epitope glycosylation, specifically a2,6 sialylation, on the binding of Pola-V to CD79B and thereby its ability to kill tumor cells. Specifically, we identify the exact glycosylated residues on CD79A and CD79B which create a glycan shield around the Pola-V binding site, precluding binding to its target. We show how genetic, pharmacological and enzymatic approaches that remove terminal sialic acid residues from these N-linked glycans lead to enhanced tumor killing by Pola-V. We hypothesize and test multiple methods of targeting this pathway in order to enhance Pola-V killing both in vitro and in vivo. Finally, we reveal a previously unappreciated role for the ubiquitin ligase KLHL6 in regulating CD79B protein abundance and surface expression of the B cell antigen receptor (BCR), including how this pathway is used by physiological germinal center B cells and how it is corrupted to enhance BCR expression in GCB DLBCL. Conclusions: These findings unravel the molecular basis of response heterogeneity to Pola-V and identify approaches that might be deployed therapeutically to enhance the efficacy of CD79B-specific tumor killing. In addition, we identify how KLHL6 determines expression of the BCR in both physiological and malignant germinal center B cells, and how KLHL6 mutation may modulate sensitivity of GCB DLBCL tumors to Pola-V.

Favezelimab (anti-LAG-3) Plus Pembrolizumab in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma (DLBCL): Cohort 3 of a Multicohort Open-Label Phase 1/2 Study

Background: Lymphocyte-activation gene 3 (LAG-3) is involved in the regulation of T-cell function and is commonly coexpressed with PD-1 on anergic T cells. Favezelimab (MK-4280), a humanized IgG4 anti-LAG-3 monoclonal antibody, plus pembrolizumab (anti-PD-1) is being investigated in the multicohort phase 1/2 MK-4280-003 efficacy and safety study (NCT03598608) in patients with relapsed or refractory (R/R) hematologic malignancies. Prior analyses of the combination demonstrated antitumor activity and manageable safety in patients with anti-PD-1-naive R/R classical Hodgkin lymphoma (cHL; cohort 1; ORR, 73%; CR, 30%) (Johnson NA et al. Blood. 2022;140(suppl 1):6540-2) and anti-PD-1-refractory cHL (cohort 2; ORR, 29%; CR, 9%) (Timmerman J et al. Blood. 2022;140(suppl 1):768-70). We present results from analysis of patients with R/R DLBCL enrolled in cohort 3. Methods: In this study, a safety lead-in phase (part 1) to determine the recommended phase 2 dose (RP2D) was followed by a dose-expansion phase (part 2). In cohort 3, eligible patients were ≥18 years old, had histologically confirmed R/R DLBCL that had progressed after ≥2 lines of previous therapy, including progression after autologous stem cell transplant (ASCT), had declined ASCT, or were ineligible for ASCT. Patients with Richter transformation were not permitted. In part 1, patients received favezelimab at a starting dose of 200 mg that was escalated to 800 mg plus pembrolizumab at 200 mg IV every 3 weeks (Q3W) per the modified toxicity probability interval method. In the dose-expansion phase, patients received favezelimab at the established RP2D of 800 mg plus pembrolizumab 200 mg Q3W for ≤35 cycles (~2 years). Response assessments were performed at weeks 12 and 24 (PET) and Q12W (CT). Adverse events (AEs) were graded per the NCI CTCAE v4.0. The primary end point was safety. ORR per IWG 2007 criteria by investigator review was a secondary end point. Exploratory end points included duration of response (DOR) and progression-free survival (PFS) per IWG 2007 criteria by investigator review and overall survival (OS). Results: A total of 25 patients with R/R DLBCL were enrolled. Patients had a median age of 73 years (range, 25-87), 10 (40%) had ECOG PS 0, and 15 (60%) had ≥3 prior lines of therapy. Most common subtypes of DLBCL were unspecified DLBCL (n = 12; 48%) and germinal center B-cell DLBCL (n = 6; 24%). At database cutoff (March 2, 2023), 1 patient (4%) was ongoing on treatment and 24 (96%) had discontinued because of progressive disease (n = 14), AEs (n = 2), clinical progression (n = 5), or patient noncompliance/nonstudy anticancer therapy (n = 3); 1 patient (4%) discontinued because of treatment-related AEs. No treatment-related deaths occurred. Sixteen patients (64%) had a treatment-related AE; the most common (≥5%) were cough (16%), increased blood alkaline phosphatase (12%), and hypothyroidism, constipation, infusion related reaction, increased AST, muscle spasms, headache and pruritis (8% each). Grade 3 or 4 treatment-related AEs occurred in 4 patients (16%; 1 grade 3 lymphocytic hypophysitis, 1 grade 3 infectious enterocolitis, 1 grade 3 increased AST, 1 grade 3 increased ALT, 1 grade 3 increased amylase, and 1 grade 4 decreased neutrophil count). AEs of clinical interest occurred in 5 patients (20%); only one grade ≥3 occurred (grade 3 hypophysitis). The median (range) time from first dose to data cutoff was 25.9 (19.7-52.1) months. The objective response rate was 12% (95% CI, 2.5-31.2; [2 CR, 1 PR]). Among 17 patients with a postdose scan, 7 (41%) had a reduction from baseline in target lesion size, and 6 (35%) had ≥50% reduction from baseline. Median DOR was not reached (range, 3.0+ to 16.9+ months); 1 responder had an observed response duration of ≥12 months. Median PFS was 2.1 months (95% CI, 1.1-2.7); 12-month PFS rate was 12%. As of the data cutoff, 20 patients (80%) had died. Median OS was 6.4 months (95% CI, 2.3-15.8); 12-month OS rate was 43%. Conclusion: Favezelimab 800 mg plus pembrolizumab 200 mg had limited antitumor activity in patients with DLBCL in cohort 3. Analyses are underway to identify biomarkers predictive of response to the combination of favezelimab and pembrolizumab. The safety profile was manageable and consistent with that observed in other cohorts in the study.

The altered metabolic pathways of diffuse large B-cell lymphoma not otherwise specified

Altered metabolic fingerprints of Diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS) may offer novel opportunities to identify new biomarkers and improve the understanding of its pathogenesis. This study aimed to investigate the modified metabolic pathways in extranodal, germinal center B-cell (GCB) and non-GCB DLBCL NOS from the head and neck. Formalin-fixed paraffin-embedded (FFPE) tissues from eleven DLBCL NOS classified according to Hans’ algorithm using immunohistochemistry, and five normal lymphoid tissues (LT) were analyzed by high-performance liquid chromatography-mass spectrometry-based untargeted metabolomics. Partial Least Squares Discriminant Analysis showed that GCB and non-GCB DLBCL NOS have a distinct metabolomics profile, being the former more similar to normal lymphoid tissues. Metabolite pathway enrichment analysis indicated the following altered pathways: arachidonic acid, tyrosine, xenobiotics, vitamin E metabolism, and vitamin A. Our findings support that GCB and non-GCB DLBCL NOS has a distinct metabolomic profile, in which GCB possibly shares more metabolic similarities with LT than non-GCB DLBCL NOS.

The RNA helicase DDX21 cooperates with ETS1 and FLI1 in cell cycle regulation and small nucleolar RNA processing to sustain the survival of DLBCL cells

Introduction: We have previously shown that the two ETS transcription factors ETS1 and FLI1, co-mapped in the 11q24.3 region, are recurrently gained in up to 25% of diffuse large B cell lymphomas (DLBCL), and largely co-regulate a series of genes involved in B cell signaling, differentiation and cell cycle (Bonetti et al., 2013; Priebe et al., 2020; Sartori et al., 2021). ABC-DLBCL is a more aggressive subtype of DLBCL compared to GCB-DLBCL, and it is associated with poor outcomes when treated with a standard therapy. As a result, there is an urgent need to elucidate new therapeutic venues for this dismal malignancy. While FLI1 is expressed at a higher level in DLBCL of the germinal center B-cell (GCB) type than in the activated B-cell-like (ABC) DLBCL, ETS1 is more expressed in the latter subgroup. We and others have reported preclinical anti-tumor activity in lymphomas and other tumors with small molecules blocking the binding of ETS factors and RNA helicases (Erkizan et al., 2009; Spriano et al., 2019). In this study, we identified additional therapeutic targets related to these transcription factors, by investigating the ETS1 interactome in ABC DLBCL. Methods: Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS) was done on proteins obtained with pull down of strep-tagged ETS1. Proteins with a spectral count above 4 were considered candidate interactors, and validated by normal and reverse co-immunoprecipitation experiments. RNASeq and small RNASeq analysis was done after DDX21 siRNAs silencing in 2 ABC (HBL1 and U2932) and in 2 GCB (OCI-Ly1 and VAL) DLBCL cell lines. In addition, we performed ChIPSeq for DDX21 on the same 4 cell line models. Results: Proteins related to RNA processing, more specifically in spliceosome (NOP56 and ALYREF) and in ribosome biogenesis (SF3B1 and DDX21), were among the identified ETS1 interactors in the ABC DLBCL HBL-1 cell line. We focused on the novel ETS1 interactor DDX21, an RNA helicase also regulated by FLI1 (Sartori et al., 2021). DDX21 appeared more expressed in ABC than GCB DLBCL (P < 0.001) in 4 datasets (GSE98588, n = 117; phs001444.v2.p1, n = 432; GSE95013, n = 33; GSE10846, n = 350). When we silenced DDX21 with siRNAs, toxicity was seen in ABC (U2932) and not in GCB (OCI-Ly1) cell lines. Our results indicate DDX21 is involved in regulating proteins involved in cell cycle (FDR < 0.001), ribosomes (FDR < 0.001), spliceosome (FDR < 0.001) and small nucleolar RNAs (snRNAs). Conclusions: In ABC DLBCL, ETS1 interacts with proteins involved in spliceosome and in ribosome biogenesis, including DDX21. Highly expressed in ABC than GCB DLBCL, DDX21 sustains the survival of lymphoma cells by regulating cell cycle and RNA processing. Targeting the interaction between ETS1 and DDX21 represents a novel therapeutic modality against ABC DLBCL. The research was funded by: Swiss Cancer Research grant KLS-3580-02-2015 (to FB) and by Rotary Foundation grants GG1639200 and GG1756935 (to GS) Keywords: Aggressive B-cell non-Hodgkin lymphoma, Genomics, Epigenomics, and Other -Omics, Molecular Targeted Therapies No conflicts of interests pertinent to the abstract.

Non-germinal center B-cell subtype of pediatric diffuse large B-cell lymphoma in Japan: A retrospective cohort study.

Diffuse large B-cell lymphoma (DLBCL) is classified into two molecular subtypes according to its cell of origin: germinal center B-cell (GCB) subtype and activated B-cell/non-GCB subtype. This latter subtype shows a poorer prognosis in adults. However, in pediatric DLBCL, the prognostic impact of the subtype is yet to be clarified. This study sought to compare the prognosis between GCB and non-GCB DLBCL in a large number of cases in children and adolescents. In addition, this study intended to describe the clinical, immunohistochemical, and cytogenetic characteristics of these two molecular subtypes of DLBCL, and consider differences in the biology, frequency, and prognosis of GCB and non-GCB subtypes in pediatric versus adult DLBCL or in Japanese versus Western pediatric DLBCL patients. We selected mature B-cell lymphoma/leukemia patients for whom specimens had been submitted to the central pathology review in Japan between June 2005 and November 2019. We referred the past studies on Asian adult patients and Western pediatric patients to compare with our results. Data were obtained from 199 DLBCL patients. The median age of all patients was 10years, with 125 patients (62.8%) in the GCB group and 49 (24.6%) in the non-GCB group other than 25 cases whose immunohistochemical data were insufficient. Overall, the percentage of translocation of MYC (1.4%) and BCL6 (6.3%) was lower than in adult and Western pediatric DLBCL cases. The non-GCB group showed a significantly higher proportion of females (44.9%), a higher incidence of stage III disease (38.8%), and B-cell lymphoma 2 (BCL2)-positivity in immunohistochemistry (79.6%) compared to the GCB group; however, no BCL2 rearrangement was observed in both GCB and non-GCB groups. The prognosis did not differ significantly between the GCB and non-GCB groups. This study including a large number of non-GCB patients showed the same prognosis between GCB and non-GCB groups and suggested a difference in the biology of pediatric and adolescent DLBCL compared to adult DLBCL as well as between Asian and Western DLBCL.

The Composition, Cell Interaction Patterns and Prognostic Impact of the Tumor Microenvironment in Germinal Center B-Cell and Activated B-Cell-like Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma not otherwise specified (DLBCL NOS) is divided into germinal center B-cell (GCB) and activated B-cell-like (ABC) molecular subtypes, which differ both biologically and clinically, ABC DLBCL often presenting with less favorable outcome. The clinical course of DLBCL is also impacted by the tumor microenvironment (TME). However, differences in the composition of the TME between the GCB and ABC DLBCLs have not been uncovered. Materials and Methods: We analyzed gene expression from 81 primary DLBCL samples using the Nanostring nCounter Human PanCancer Immunoprofiling Panel, and characterized B cells (CD20), T cells (CD3, CD4, CD8, FOXP3, TBET, Granzyme B (GrB)), macrophages (CD68, CD163), NK cells (CD56, GrB), and immune checkpoint molecules expressed on these cells (PD-1, PD-L1, TIM3) from 178 tumors using multiplex immunohistochemistry (mIHC). In addition, we measured HLA-ABC, HLA-DR, and B2M expression immunohistochemically. Finally, we performed cell-cell interaction analyses (Brück et al. Mod Pathol. 2021). The patients were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP)-like immunochemotherapy, and the findings correlated with patient demographics and survival. Results: As expected, differential gene expression analysis between the molecular subtypes revealed that ABC DLBCLs expressed significantly higher levels of TNFRSF13B, IRF4 and AICDA. In addition, genes related to macrophage signaling, such as CD163, MARCO and CD47 were upregulated. In contrast, GCB DLBCLs expressed higher levels of genes associated with T cell receptor signaling, such as ICOSLG, HLA-DOB, IL4 and CD40LG. Based on mIHC (Figure 1A), CD163+ tumor associated macrophages (TAMs) were enriched in the ABC DLBCL TME (adj. p < 0.001), whereas in the GCB DLBCL TME, TAMs were more often CD163- (adj. p < 0.001). In the GCB DLBCL TME, a higher proportion of all tumor-infiltrating T cells were T helper cells (CD4+; adj. p = 0.023), and regulatory T cells (FOXP3+; adj. p = 0.026), while GrB+ cytotoxic cells were more abundant in the ABC subtype (adj. p = 0.006). Consistently, ABC DLBCLs were more frequently HLA-ABC and B2M positive compared to GCB DLBCLs (p = 0.005 and p = 0.002, respectively). Interestingly, in ABC DLBCL, CD163+ TAMs interacted more often with T cells, other TAMs, and B cells than in GCB DLBCL (adj. p < 0.001 for all). Instead, in GCB DLBCL, T cells shared more interactions with other T cells and macrophages (adj. p < 0.001 for all). However, while T cells had generally more interactions in GCB DLBCL, cytotoxic T cells had more cell interactions in ABC DLBCL (adj. p < 0.001). The prognostic impact on tumor-infiltrating immune cells on survival varied between the two molecular subtypes (Figure 1B). High proportion of immune checkpoint-expressing TAMs associated with unfavorable survival in both molecular subtypes. However, in the patients with GCB DLBCL, high proportion of PD-L1+, TIM3+ and GrB+ cells predicted unfavorable survival, whereas in the patients with ABC DLBCL, high proportion of CD8+ cytotoxic T cells associated with favorable outcome. Conclusions: The TME of GCB and ABC-like DLBCL are distinct by composition, cell-cell interaction patterns and in terms of prognostic biomarkers. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Functional Profiling Identifies Selective Enhancer Dependencies in MYC-Intact and MYC-Rearranged Diffuse Large B-Cell Lymphoma

Strong expression of MYC and MYC-regulated genes defines the molecular high-grade / double-hit signature subgroup of germinal center B-cell diffuse large B-cell lymphoma (GCB-DLBCL) which shows poor clinical outcomes with current therapy. About half of such cases show large-scale chromosomal rearrangements between the MYC locus and heterologous enhancer-bearing loci, but there is little understanding of the specific regulatory elements that activate MYC in absence of a rearrangement, or in the setting of non-immunoglobulin MYC rearrangement partners (RPs). We used high-throughput CRISPR-interference functional profiling (CRISPRi) to pinpoint cis-regulatory element dependencies in MYC-intact and MYC-rearranged lymphoma cell lines at the level of individual nucleosome-free regions (NFRs). Custom sgRNA libraries targeted 233 individual NFRs (median 42 sgRNA / NFR after stringent quality filtering) across the MYC topological domain (TAD) and MYC RP loci in six GCB-DLBCL cell lines and two comparator mantle cell lymphoma (MCL) cell lines. Promoter-targeting sgRNAs against 587 sequence-specific transcription factor (TF) genes and 65 essential gene controls allowed for interrogation of trans-factor dependencies in the same assay. CRISPRi profiling in one GCB-DLBCL and one MCL cell line with intra-chromosomal rearrangements of chromosome 8 showed strong, unique dependencies on enhancers within the cell-line-specific RPs, supporting an "enhancer-hijacking" function of these rearrangements. Three profiled cell lines showed structurally distinct variants of a recurrent t(3;8)(q27;q24) rearrangement that links the MYC gene to the distal super-enhancer region 150-240 kB upstream of BCL6. Although the distal BCL6 super-enhancers have been shown to sustain the essential BCL6 oncogene itself, we surprisingly found only weak dependency on the distal BCL6 enhancers for the three GCB-DLBCL cell lines lacking t(3;8) rearrangements. In contrast, all three GCB-DLBCL cell lines with t(3;8) rearrangements showed strong dependency on specific NFRs within the distal BCL6 super-enhancer. Validation assays showed that concurrent targeting of two BCL6 enhancer NFRs lead to even greater growth suppression and loss of MYC expression in t(3;8)-bearing cell lines. In both GCB-DLBCL cell lines lacking MYC rearrangements, we identified a strong, novel dependency on a specific enhancer ("GCB-ME1") in the 3' portion of the MYC TAD. This enhancer was acetylated in MYC-intact GCB-DLBCL patient samples and normal centroblasts, but not in MCL or MYC-rearranged DLBCL samples. We confirmed that sustained MYC expression in MYC-intact cell lines requires activity of GCB-ME1. GCB-ME1 showed little or no essentiality in the four MYC-rearranged DLBCL cell lines or in the MYC-intact MCL cell line SP-49, which is dependent on previously identified Notch/RBPJ-regulated 5' MYC enhancers (Ryan et al., 2017). ChIP-Seq showed that both GCB-ME1 and all of the most strongly essential BCL6 enhancers in t(3;8) cell lines were strongly bound by a triad of TFs, MEF2B, POU2F2, and POU2AF1, that were previously implicated in BCL6 activation. These genes were required to sustain MYC expression in both MYC-intact and t(3;8) GCB-DLBCL, and were highly essential in those cell lines, but POU2F2 and MEF2B were not essential in some cell lines with alternate MYC rearrangements. Our data show that DLBCL is highly sensitive to selective disruption of discrete MYC-activating enhancers. Transcriptional regulatory factor dependencies in DLBCL may be shaped by the requirement to sustain MYC activation via the native GCB-ME1 enhancer or alternately via enhancers in specific RP loci, a finding with implications for therapeutic targeting of transcriptional regulatory mechanisms in DLBCL. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antigen-Independent, Autonomous B-Cell Receptor Signaling in Diffuse Large B-Cell Lymphoma

Gene expression profiling of diffuse large B-cell lymphoma (DLBCL) defines the Cell-of-origin (CoO) classification with two major subtypes, germinal center B-cell (GCB) and the more aggressive activated B-cell (ABC) type. Chronic activation of B-cell receptor (BCR) signaling with constitutive activation of the NF-κB pathway is an essential oncogenic mechanism in ABC DLBCL. BCR pathway activation with increased NF-κB activation can be caused by gain-of-function mutations of the BCR signaling cascade. We investigated whether antigen-independent, i.e. autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia, acts as a functional driver mechanism in DLBCL without such gain-of-function mutations. DNA and RNA were extracted from ABC DLBCL cell lines TMD8 and OCI-Ly3 and from fresh-frozen biopsies of 18 histologically confirmed DLBCL cases. Functional BCR transcripts were identified by ARTISAN PCR and transduced into murine triple knock out (TKO) cells. TKO cells lack the recombination activation gene 2 (Rag2) and lambda 5 gene (λ5) and are therefore incapable of expressing an endogenous BCR. In addition, the BCR signal transduction molecule SLP65 is replaced with a tamoxifen-inducible estrogen receptor (ERT2) - SLP65 fusion gene. Upon transduction of both heavy and light BCR chains and staining with Indo-1, background calcium flux can be measured as Indo-1 signal intensity ratio (SIR) by flow cytometry. After restoring functionality of the BCR signaling cascade by tamoxifen, antigen-independent BCR signaling is measured as SIR, followed by maximum BCR stimulation with an isotype-specific crosslinking antibody. Autonomous BCR signaling strength (SSBCR) is quantified by an arithmetic product based on the relative Indo-1 SIR and the relative number of cells with SIR above background in the presence of tamoxifen and calibrated on crosslinking-induced maximum SIR. According to the primary hypothesis, the BCR of CARD11 wild-type, IgM-expressing TMD8 cells demonstrated strong autonomous BCR signaling (SSBCR=0.243), whereas the BCR of CARD11 mutated, IgG-expressing OCI-Ly3 cells completely lacked autonomous signaling (SSBCR=0.022). BCR from 13 primary DLBCL signaled autonomously (SSBCR=0.125-0.619), where 5 samples were negative (SSBCR=0.004-0.023) Autonomous BCR signaling was absolutely dependent on IgM isotype (Fisher's exact test: p=0.0016). CoO classification by digital gene expression (Nanostring) analyses or immunohistochemistry demonstrated autonomous BCR signaling in 8/9 ABC (non-GCB) DLBCL, 2/6 GCB DLBCL, and 2 unclassifiable DLBCL, and a transformed follicular lymphoma DLBCL. Independent of CoO, DLBCL can be assigned to 6-7 clusters based on their genetic aberrations (Chapuy 2018, Wright 2020). Fifteen DLBCL were suited for WES and targeted panel sequencing. Four cases were assigned by a probabilistic calculation to Chapuy cluster C5, 2 cases to C2, and one case to C1, while 8 cases remained unclassifiable. Clustering by the LymphGen tool assigned 3 cases to cluster ST2, one case each into MCD, EZB, and N1, 4 cases into various clusters, and 5 cases unclassified. DLBCL with autonomous BCR signaling fell into clusters C1/unclassified, C2/unclassified, C5/unclassified (n=3), unclassified/EZB, unclassified/ST2, unclassified/N1 and unclassified/unclassified (n=2). Cases lacking autonomous BCR signaling were assigned to unclassified/ST2 (n=2), C5/MCD, and unclassified/unclassified. Eleven of 13 cases with an autonomous signaling BCR lacked activating CARD11 mutations. In conclusion, autonomous BCR signaling is a potential immunological oncogenic driver in DLBCL that cannot be detected by sequencing studies, is independent of external antigen stimulation, and originates from a self-organizing conformation of the surface BCR (Minici 2017). Autonomous BCR signaling is present predominantly in ABC DLBCL and dependent on IgM isotype. Autonomous BCR signaling offers a convincing explanation for oncogenic NF-κB activation in DLBCL. Limited data suggest that the majority of DLBCL with autonomous BCR signaling fall into the ABC-dominated genetic clusters C1/C5, and MCD/N1, respectively. In summary, genetic aberrations, BCR function, and gene expression profile form 3 independent layers of DLBCL pathophysiology that need to be analyzed separately and offer independent targets for therapy.

TPL2, a New Prognostic Factor and a Potential Therapeutic Target in ABC-DLBCL

Background: Activated B-cell like diffuse large B-cell lymphoma (ABC-DLBCL) is a subtype of DLBCL, characterized by the constitutive activation of NF-κB and JAK/STAT3 pathways on the background of the chronic active BCR signaling. Being featured by mutations in MYD88 and/or CD79B, it potentially comprises the MCD/C5 subtype in the recent genetics-based categorization. In light of the inferior prognosis of this subtype to germinal center B-cell (GCB) like DLBCL, identification of subtype-specific strategies has been sought. Tumor progression locus 2 (TPL2) is an intracellular serine/threonine kinase that is reported to be activated downstream of TLRs/MYD88 signaling and to play roles in oncogenesis. However, the involvement of TPL2 in B-cell malignancies is less defined despite relatively higher transcript expression than in solid tumors. Results: To address the impact of TPL2 protein levels on DLBCL phenotypes, we performed an IHC analysis on 79 FFPE samples of DLBCL diagnosed and treated at our institution (48 non-GCB and 31 GCB according to Hans algorithm). When divided into two groups, one with strong TPL2 expression (TPL-high) and the other with no/weak expression (TPL2-low), the ratio of TPL2-high did not differ between non-GCB and GCB (67% vs 64%). However, TPL2-high had a significantly higher percentage of positive B symptom (42% vs 11%, p=0.005) and extra-nodal involvement (75% vs 52%, p=0.047) compared to TPL2-low, and these trends were observed more specifically in non-GCB. Moreover, it is noteworthy that TPL2-high showed a significantly lower overall survival in non-GCB (p=0.037), but did not in GCB (p=0.999) (Figure 1). Intriguingly, the analysis combined with pSTAT3 (Y705) staining revealed that TPL2-high accompanied by positive pSTAT3 tended to display particularly poor prognosis in non-GCB. Then, we analyzed the mechanism by which TPL2 is involved in the tumorigenesis of ABC-DLBCL using TMD8, a well-described ABC-DLBCL cell line with MYD88L265P and CD79BY196 mutations. A specific TPL2 inhibitor significantly affected the cell proliferation of TMD8 cells, whereas did not on the cells with GCB phenotype. Of note, TPL2 inhibitor induced G1 cell-cycle arrest via downregulation of CyclinE and CDK2 and upregulation of p27 in western blot analysis, which was confirmed by quantitative PCR. Next, we investigated the downstream signaling of TPL2 in ABC-DLBCL. Western blot analysis revealed that TPL2 inhibitor as well as genetic TPL2 knock down suppressed the activation of NF-κB components and STAT3, and the expression of c-MYC. These results implied the involvement of TPL2 in IL-6/IL-10 autocrine loop and resultant JAK/STAT3 activation. As expected, treatment with TPL2 inhibitor downregulated IL-6/IL-10 mRNA by quantitative PCR and accordingly decreased the levels of IL-6/IL-10 in the cell culture supernatant of treated TMD8 cells. Therefore, we speculated that TPL2 promoted the oncogenic activation of NF-κB and STAT3 pathways via IL-6/IL-10 upregulation in ABC-subtype. Lastly, we evaluated the physiological function of TPL2 by western blot analysis using 293T. Although an overexpression TPL2 alone did not elicit NF-κB and STAT3 activation, when co-expressed with MYD88L265P TPL2 obviously activated these pathways, whereas dominant negative TPL2 failed to demonstrate those effects even with MYD88L265P co-expression. These findings indicated that TPL2 could possibly exert its tumorigenicity in MYD88L265P-dependent manner in ABC-DLBCL. Conclusion: In association with the importance as an unfavorable prognostic factor, our results suggested that TPL2 might mediate the crosstalk between the oncogenic MYD88L265P and the activation of NF-κB and STAT3 pathways, coordinately contributing to the tumorigenesis of ABC-DLBCL. From this standpoint, TPL2 could emerge as a new therapeutic target in ABC-DLBCL. Figure legend Overall survival of non-GCB (A) and GCB (B) DLBCL patients, with regard to the TPL2 expression: TPL2-low (red line) or TPL2-high (black line). Overall survival was determined by the Kaplan-Meier method and compared by using the log-rank test. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 741: Evaluation of etiologic heterogeneity for risk of diffuse large B-cell lymphoma (DLBCL) subtype defined by cell-of-origin

Abstract Background: DLBCL is the most common non-Hodgkin lymphoma subtype in western countries and is clinically heterogeneous. Gene expression profiling has identified two major biologically distinctive DLBCL subtypes defined by their cell-of-origin (COO): germinal center B-cell (GCB) - characterized by BCL2 rearrangement and C-REL amplification, and activated B-cell (ABC) - characterized by constitutive activation of the NF-kB pathway. We evaluated putative DLBCL risk factors for etiologic heterogeneity as defined by COO. Methods: We used a clinic-based study of newly diagnosed NHL cases and frequency matched controls, enrolled from 2002-2014, with a total of 687 DLBCL cases and 2253 controls for this analysis. Using formalin-fixed, paraffin-embedded tumor tissue, we determined COO by either digital expression profiling (NanoString; classified as GCB, ABC, undetermined) or clinically using the Hans algorithm (immunohistochemical markers; classified as GCB, non-GCB, undetermined). Integrating these two sources of COO data, there were 271 GCB, 170 non-GCB, and 246 undetermined/missing (mainly due to lack of tissue) cases. Risk factor data were collected from self-administered questionnaires and included family history of hematologic malignancy; history of atopy, allergy, asthma, eczema, or autoimmune disease; blood transfusion; vaccination history; regular low-dose aspirin use; body mass index (BMI) 2 years prior diagnosis/enrollment; smoking history; alcohol consumption (never/current/former); leisure-time physical activity; and recreational sun exposure. Polytomous logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals for COO subtypes, adjusted for age, sex and residence. Results: The median age of cases was 64 years with 55% male; the median age of controls was 64 years and 53% male. The association with BMI (30+ vs. 18.5-24.9 kg/m2) was stronger for non-GCB (OR=2.21; 1.40-3.49) than for GCB (OR=1.70; 1.21-2.40) DLBCL (P-heterogeneity 0.02), while a family history of hematologic malignancy was associated with non-GCB (OR=2.27; 1.47-3.50) but not GCB (OR=1.30; 0.86-1.96) DLBCL, although the P-heterogeneity was not significant (P=0.13). For GCB, there was an inverse association with former (vs. never) alcohol use for GCB (OR=0.57; 0.39-0.83) but not for non-GCB (OR=0.98; 0.61-1.57) DLBCL (P-heterogeneity 0.007), and an inverse association with regular use of low-dose aspirin for GCB (OR=0.64; 0.47-0.87) but not for non-GCB (OR=0.88; 0.61-1.25) DLBCL (P-heterogeneity 0.05). No other risk factors evaluated showed important heterogeneity by COO. Conclusions: These initial results suggest most DLBCL risk factors do not show etiologic heterogeneity by DLBCL COO, although associations with BMI and family history with non-GCB DLBCL and alcohol and low-dose aspirin use with GCB DLBCL warrant follow-up. Citation Format: Geffen Kleinstern, Dennis P. Robinson, Lisa M. Rimsza, Melissa C. Larson, Rebecca L. King, Grzegorz S. Nowakowski, Carrie A. Thompson, Stephen M. Ansell, Matthew J. Maurer, Andrew L. Feldman, Susan L. Slager, Anne J. Novak, Thomas M. Habermann, James R. Cerhan. Evaluation of etiologic heterogeneity for risk of diffuse large B-cell lymphoma (DLBCL) subtype defined by cell-of-origin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 741.

Reproducibility of Gene Expression Signatures in Diffuse Large B-Cell Lymphoma.

Simple SummaryMultiple gene expression signatures with biological or prognostic subgroups have been published in diffuse large B-cell lymphoma (DLBCL). With exception of the cell of origin (COO) classifier, these were not validated in independent cohorts. The aim of the study was to reproduce four gene expression signatures capturing multiple biological subgroups using the NanoString platform. In addition, we aimed to identify potential associations between the signatures and portray the heterogeneity of DLBCL. We show that, in an independent cohort of clinically well-defined patients, these signatures can co-occur in the same patient and that each classifier captures a different aspect of the biological heterogenous panorama of DLBCL. Beside COO, there is clear evidence of different immune and MYC signatures. A direct comparison in our cohort showed that these signatures reflect independent biological features. More comparative studies with gene expression profiles need to be conducted to enable a further integration and to help develop new taxonomy systems for clinical utility.Multiple gene expression profiles have been identified in diffuse large B-cell lymphoma (DLBCL). Besides the cell of origin (COO) classifier, no signatures have been reproduced in independent studies or evaluated for capturing distinct aspects of DLBCL biology. We reproduced 4 signatures in 175 samples of the HOVON-84 trial on a panel of 117 genes using the NanoString platform. The four gene signatures capture the COO, MYC activity, B-cell receptor signaling, oxidative phosphorylation, and immune response. Performance of our classification algorithms were confirmed in the original datasets. We were able to validate three of the four GEP signatures. The COO algorithm resulted in 94 (54%) germinal center B-cell (GCB) type, 58 (33%) activated B-cell (ABC) type, and 23 (13%) unclassified cases. The MYC-classifier revealed 77 cases with a high MYC-activity score (44%) and this MYC-high signature was observed more frequently in ABC as compared to GCB DLBCL (68% vs. 32%, p < 0.00001). The host response (HR) signature of the consensus clustering was present in 55 (31%) patients, while the B-cell receptor signaling, and oxidative phosphorylation clusters could not be reproduced. The overlap of COO, consensus cluster and MYC activity score differentiated six gene expression clusters: GCB/MYC-high (12%), GCB/HR (16%), GCB/non-HR (27%), COO-Unclassified (13%), ABC/MYC-high (25%), and ABC/MYC-low (7%). In conclusion, the three validated signatures identify distinct subgroups based on different aspects of DLBCL biology, emphasizing that each classifier captures distinct molecular profiles.

SGK1 mutation status can further stratify patients with germinal center B-cell-like diffuse large B-cell lymphoma into different prognostic subgroups.

There are over a 100 driver gene mutations in patients with diffuse large B‐cell lymphoma (DLBCL), but their clinical significance remains unclear. Here, we first analyzed the DLBCL dataset from the UK‐based Haematological Malignancy Research Network. Patients were divided into high‐ and low‐risk groups based on whether lymphoma progressed within 24 months. Genes showing significantly different frequencies between groups were selected. Survival data for patients with the selected mutant genes were analyzed. The results were validated using two other large databases to evaluate the relationship between the selected mutant genes and prognosis. The mutation frequencies of 11 genes (MYD88[L265P], SGK1, MPEG1, TP53, SPEN, NOTCH1, ETV6, TNFRSF14, MGA, CIITA, and PIM1) significantly differed between the high‐ and low‐risk groups. The relationships between these mutant genes and patient survival were analyzed. Patients who harbored SGK1 (serum and glucocorticoid‐inducible kinase 1) mutations exhibited the best prognosis. Most patients with SGK1 mutation are germinal center B‐cell (GCB) subtype. Among patients with GCB DLBCL, those harboring SGK1 mutations exhibited better prognosis than those without SGK1 mutations. Most SGK1 mutations were single‐base substitutions, primarily scattered throughout the catalytic domain‐encoding region. Multiple SGK1 mutations were identified in a single patient. Thus, SGK1 mutations are a marker of good prognosis for DLBCL and occur predominantly in the GCB subtype of DLBCL. SGK1 mutation status can further stratify patients with GCB DLBCL into different prognostic subgroups.

ASB2 is a direct target of FLI1 that sustains NF-\u03baB pathway activation in germinal center-derived diffuse large B-cell lymphoma

BackgroundDiffuse large B-cell lymphoma (DLBCL) comprises at least two main biologically distinct entities: germinal center B-cell (GCB) and activated B-cell (ABC) subtype. Albeit sharing common lesions, GCB and ABC DLBCL present subtype-specific oncogenic pathway perturbations. ABC DLBCL is typically characterized by a constitutively active NF-kB. However, the latter is seen in also 30% of GCB DLBCL. Another recurrent lesion in DLBCL is an 11q24.3 gain, associated with the overexpression of two ETS transcription factors, ETS1 and FLI1. Here, we showed that FLI1 is more expressed in GCB than ABC DLBCL and we characterized its transcriptional network.MethodsGene expression data were obtained from public datasets GSE98588, phs001444.v2.p1, GSE95013 and GSE10846. ChIP-Seq for FLI1 paired with transcriptome analysis (RNA-Seq) after FLI1 silencing (siRNAs) was performed. Sequencing was carried out using the NextSeq 500 (Illumina). Detection of peaks was done using HOMER (v2.6); differential expressed genes were identified using moderated t-test (limma R-package) and functionally annotated with g:Profiler. ChIP-Seq and RNA-Seq data from GCB DLBCL cell lines after FLI1 downregulation were integrated to identify putative direct targets of FLI1.ResultsAnalysis of clinical DLBCL specimens showed that FLI1 gene was more frequently expressed at higher levels in GCB than in ABC DLBCL and its protein levels were higher in GCB than in ABC DLBCL cell lines. Genes negatively regulated by FLI1 included tumor suppressor genes involved in negative regulation of cell cycle and hypoxia. Among positively regulated targets of FLI1, we found genes annotated for immune response, MYC targets, NF-κB and BCR signaling and NOTCH pathway genes. Of note, direct targets of FLI1 overlapped with genes regulated by ETS1, the other transcription factor gained at the 11q24.3 locus in DLBCL, suggesting a functional convergence within the ETS family. Positive targets of FLI1 included the NF-κB-associated ASB2, a putative essential gene for DLBCL cell survival. ASB2 gene downregulation was toxic in GCB DLBCL cell lines and induced NF-κB inhibition via downregulation of RelB and increased IκBα. Additionally, downregulation of FLI1, but not ASB2, caused reduction of NF-κB1 and RelA protein levels.ConclusionsWe conclude that FLI1 directly regulates a network of biologically crucial genes and processes in GCB DLBCL. FLI1 regulates both the classical NF-κB pathway at the transcriptional level, and the alternative NF-κB pathway, via ASB2. FLI1 and ASB2 inhibition represents a potential novel therapeutic approach for GCB DLBCL.

Combination Benefit of Capivasertib and Venetoclax in Preclinical Models of Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common B-cell malignancy among adults and despite approximately 65% of patients with DLBCL being cured with RCHOP therapy, nonresponsive and relapsed patients have inadequate treatment options, highlighting the importance for innovative treatment regimens. Blockade of B-cell receptor (BCR) downstream signaling components with various targeted agents is emerging as a clinically tractable treatment strategy across multiple B-cell malignancies. Protein Kinase B (AKT) signaling downstream of the BCR complex has been shown to be a central node in germinal center B-cell (GCB) DLBCL and the potent, selective inhibitor of AKT1, AKT2, AKT3, capivasertib, currently being evaluated in multiple clinical trials by targeting AKT-driven solid cancers, has been shown to induce apoptosis in a subset of GCB-DLBCL cell lines and cause tumor stasis in xenograft mouse models (Erdman et al., 2017). Since the monotherapy capivasertib responses in GCB DLBCL models are partial and lack durability, we hypothesized a combination approach could deliver even greater therapeutic benefit. To identify optimal partners, we conducted a capivasertib centric in vitro combination screen with specific with BH3 family members across a panel of 15 DLBCL cell lines, which revealed a synergistically active combination with the BCL2 inhibitor, venetoclax which is currently being evaluated in DLBCL. The activity was specifically enhanced in cell lines of the GCB subtype, with 4 PTEN del and 2 PTEN wt cell line models showing combination benefit. To determine the ability of this combination to drive stronger and durable responses, we assessed capivasertib and ventoclax activity in xenograft mouse models using two GCB-DLBCL cell line lines, SUDHL4 (PTEN wt) and WSU-DLCL2 (PTEN del). Oral administration of either monotherapy capivasertib (130 mg/kg BID, 4-day on/3-day off) or venetoclax (100 mg/kg QD) provided partial tumor growth inhibition (capivasertib TGI = 74% in SUDHL4 and 29% in WSU-DLCL2, and venetoclax TGI = 46% in SUDHL4 and 0% in WSU-DLCL2), whereas the combination of capivasertib and venetoclax both on a 4-day on/3-day off schedule produced complete tumor regression (100% regression) in both xenograft GCB cell line models during the dosing period. Notably, in both xenograft models all mice (5/5 per model) remained tumor free for at least 30 days following dosing cessation demonstrating high durability of response for the combination. Additionally, this combination is currently being evaluated in clinically relevant GCB and non-GCB PDX mouse models. Taken together, our results provide preclinical evidence for the rational combination of AKT and BCL-2 blockade with capivasertib and venetoclax respectively in patients with relapsed/refractory GCB-DLBCL. DisclosuresWillis: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Neveras: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Dry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mongeon: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Rosen: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Mettetal: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options. Barry: AstraZeneca: Current Employment, Other: may hold equity, stock, or stock options.

Upregulation of CD47 Expression in De Novo Diffuse Large B-Cell Lymphoma Is More Frequent in Activated B-Cell Type

CD47 is a marker of self that provides a “don't-eat-me-signal” through activation of signal-regulatory protein alpha (SIRPa), a cell surface receptor expressed on monocytes/macrophages and granulocytes. This interaction negatively regulates effector functions such as, phagocytosis, migration, and superoxide production. Upregulation of CD47 expression in cancer, including diffuse large B-cell lymphoma (DLBCL), has emerged as a mechanism to escape innate immune surveillance. Using conventional immunohistochemical detection, we assessed CD47 expression in DLBCL and interrogated its association with clinicopathologic features.Patients with de novo DLBCL were identified from two large institutions and were uniformly treated with R-CHOP and had sufficient material for study. Immunohistochemical stains (IHC) were performed on FFPE tissue (Hans algorithm, BCL2, MYC, and CD47) and scored semi-quantitatively from no reactivity (0) to strong (2 and 3; Figure 1). Mutational analysis using a 334 gene target sequencing panel, gene expression profiling using Lymph2Cx to determine the cell of origin (COO), and FISH analysis for MYC, BCL2, and BCL6 translocations, were performed. The Lymphgen tool (Wright et al, 2020) was also used to determine the DLBCL group. Fisher's exact test and Kaplan-Meier survival analysis for overall survival (OS) were performed and P <0.05 was considered significant.CD47 expression was assessed by IHC in a cohort of 152 cases of de novo DLBCL, including 107 cases of germinal center B-cell (GCB) type (70%), 37 cases of activated B-cell (ABC) type (24%), and 8 cases of intermediate type (5%). A total of 17 cases (11%) showed strong and diffuse CD47 expression with IHC scores of 2 or above (CD47hi). CD47hi cases were significantly more frequent in ABC DLBCL (24%, 9/37) than GCB DLBCL (6%, 6/107; P=0.003). The remaining 2 CD47hi cases were in the intermediate DLBCL group (25%, 2/8). ABC DLBCL with CD47hi showed more frequent mutations with TET2 (33% vs 7%; P=0.08) and ZFP36L1 (22% vs 0%; P=0.05) compared to cases with low expression of CD47 with IHC scores of less than 2 (CD47low). ABC DLBCL with CD47low showed more frequent mutations of NOTCH2 (18% vs 0%; P=0.31) and MYD88 (29% vs 11%; P=0.4) compared to CD47hi. GCB DLBCL with CD47hi showed frequent mutations of TP53 (67% vs 21%; P=0.026) and CCND1 (33% vs 0%; P=0.003) compared to CD47low. None of the 13 cases with double- or triple-hit for MYC, BCL2 and/or BCL6 showed CD47 expression. The Lymphgen tool showed that cases of DLBCL with CD47hi were mostly in the ‘other’ group (50%), with other groups represented such as ST2 (21%), EZB (14%), MCD and BN2 (1 case each). There was no difference in overall survival (OS) between CD47hi and CD47low DLBCL (5-year OS, 75% vs 72%; P=0.57), or with the GCB or ABC subtypes.Strong expression with CD47 is more frequent in ABC DLBCL and is seen in a subset of GCB DLBCL with mutations in TP53 and/or CCND1. The level of CD47 expression does not appear to predict OS in patients with DLBCL treated with R-CHOP. This study demonstrates that conventional immunohistochemical methods can readily identify DLBCL with high CD47 expression, and these patients may benefit from the use of anti-CD47 therapy. [Display omitted] DisclosuresHerrera: Gilead Sciences: Research Funding; Takeda: Consultancy; Tubulis: Consultancy; Karyopharm: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; ADC Therapeutics: Consultancy, Research Funding; Seagen: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Kite, a Gilead Company: Research Funding.

High Expression of Programmed Death-Ligand 1 (PD-L1) Correlates with Macrophage Gene Expression and Is Associated with Prolonged Progression-Free Survival (PFS) in Patients (pts) with First-Line (1L) Diffuse Large B-Cell Lymphoma (DLBCL)

▪Introduction PD-L1 and its receptor PD-1 are both expressed on multiple cell types, including antigen-presenting cells, T cells and tumor cells. In DLBCL, PD-L1 expression on non-malignant immune cells is common, whereas tumor-cell PD-L1 expression is restricted to a small percentage of pts. Because PD-L1/PD-1 are expressed on macrophages after in vitro activation, and activated macrophages contribute to antibody-dependent cellular phagocytosis (ADCP), high PD-L1 expression may identify tumors enriched with activated tumor-infiltrating macrophages primed for ADCP. Considering the role of ADCP in clinical responses to anti-CD20 (rituximab/obinutuzumab), we hypothesized that PD-L1 expression could reflect abundance of activated tumor-infiltrating macrophages and may be associated with prolonged PFS in 1L DLBCL. We leveraged 2 large Phase 3 trials and cell-line immune gene expression to better understand the biological and clinical relevance of PD-L1 expression in 1L DLBCL.Methods We used clinical, genomic and pre-treatment immunohistochemistry (IHC) data from 1L DLBCL pts treated in the MAIN (NCT00486759) and GOYA (NCT01287741) trials. Pre-treatment tissue microarray cores from 201 MAIN pts were stained for PD-L1 (clone SP263), with 21 uninvolved lymph node FFPE sections used as controls. PD-L1+ samples were defined as IHC > 1+ (> 1% tissue area occupied by PD-L1+ cells). Tumor cells were identified based on morphological/cytological features, and PD-L1 tumor expression was scored as a percentage. Activated B-cell (ABC), germinal center B-cell (GCB) and unclassified DLBCL prognostic subtypes were determined by the NanoString Lymph2Cx cell-of-origin (COO) assay. We quantified mRNA expression of PD-L1, macrophage and B-cell genes using TruSeq (Illumina) RNAseq among 702 DLBCL pts (GOYA, n = 552; MAIN, n = 150), with 28 DLBCL cell lines and 15 purified normal B-cell samples as comparators. High/low expression of each gene signature was determined by median cutoffs. Median follow-up durations were 24 and 29 mo in MAIN and GOYA, respectively (≈ 30% of PFS events in both cohorts). We used Cox regression to examine associations between these markers and PFS, adjusting for IPI, age, COO, sex and treatment arm.Results Among 201 DLBCL pts in MAIN, PD-L1 was primarily detected on inter-follicular cells with myeloid/dendritic morphology, similar to the PD-L1 staining pattern of normal lymph nodes. In samples with evaluable germinal centers (GCs), a subset of GC cells expressed PD-L1 with a punctate and granular pattern consistent with a myeloid origin. Overall, there were significantly more pts with high PD-L1 expression by IHC (IHC 3+) and mRNA among ABC vs GCB DLBCL pts. A minority of DLBCL pts (10%) had PD-L1+ tumor cells, consistent with low/undetectable CD274 (PD-L1) mRNA in the majority of DLBCL cell lines and purified normal B-cell samples tested. Additionally, among the 702 evaluable pts in both studies, CD274 mRNA inversely correlated with a B-cell gene signature (MAIN, r= −0.55; GOYA, r= −0.32). High PD-L1 expression (IHC 2+/3+) correlated with prolonged PFS (HR, 0.44 [0.24, 0.80]; P < 0.01) among the 201 evaluable pts in MAIN. Among 552 evaluable pts in GOYA, CD274 expression was higher in pts with a PET-CR (P= 0.02), particularly in pts with ABC DLBCL (P= 0.0001), with a trend toward prolonged PFS in the ABC subtype (HR, 0.80 [0.63, 1.0]; P= 0.054). CD274 mRNA positively correlated with macrophage gene expression in both cohorts (MAIN, r= 0.61; GOYA, r= 0.57); pts with high PD-L1 expression (IHC 2+/3+) also demonstrated high mRNA expression of the macrophage marker CD68. Consistent with PD-L1 reflecting abundance of activated tumor-infiltrating macrophages relevant to anti-CD20 response, high expression of a macrophage signature correlated with prolonged PFS (HR, 0.63 [0.45, 0.87]; P= 0.003) in GOYA and a trend toward prolonged PFS among DLBCL pts treated in MAIN (HR, 0.64 [0.35, 1.16]; P= 0.10), although statistical significance was not reached.Conclusions In 1L DLBCL, most pts expressed PD-L1 on immune cells with myeloid/dendritic morphology, potentially reflecting activated macrophages primed for ADCP and supportive of the correlation between high PD-L1 expression and improved clinical outcomes among pts treated with immunochemotherapy. In the context of anti-CD20, the biology of PD-L1 expression on macrophages and the impact of PD-L1/PD-1 blockade on ADCP merit further study. DisclosuresMcCord:Genentech, Inc.: Employment. Bolen:Genentech: Employment, Equity Ownership. Koeppen:Genentech, Inc.: Employment. Kadel:Genentech, Inc.: Employment. Fingerle-Rowson:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Oestergaard:F. Hoffmann-La Roche Ltd: Employment. Venstrom:Genentech, Inc.: Employment.

The Humanized EBV-LPD Recaptilates Human Non GC DLBCL

Several mouse models have been developed for diffuse large B cell lymphoma (DLBCL) such as Emu-cmyc Transgenic mice, BCL6 knock in mice, λ-myc mice and so on.However, the histopathology of these mice does not fully recapitulate that of the human DLBCL. For non- GC type in particular, it is not an exaggeration to say that none of these mice show the similarity to it.The xenograft model in which human tumor cells are transplanted into immunodeficient mice which is frequently used for the pre-clinical study for new drug, is also problematic in that the tumor cells derive form human while the cancer microenvironment from the mouse.We established the new mouse model which well recaptilates human non GC DLBCL. Human umbilical cord blood was transplanted into NOD/Shi-scid-IL2Rγnull mice(NOG mice), which is called as humanized NOG mice. The humanized NOG mice are infected with low dose EBV that infects only the primates. These mice develop EBV+ lymphoproliferative disease (LPD). We analyzed the histopathology of the LPD and compared with EBV+ DLBCL.Both EBV positive tumor cells are Mum 1 positive Bcl 6 negative CD 10 negative corresponding to non-GC DLBCL. Both tumor microenvironments showed massive infiltrate of CD 163 positive macrophages. (Figure) The localization of the cells which express cytokines such as IFN beta, TNF and IL-10 are overlapped between them.Particularly, the LPD which EBV infected humanized mice develops consists of not only EBV infected human CD20 positive cells but also human stromal cells such as human CD163 positive macrophages.In conclusion, the LPD developed in EBV infected humanized mice well recapitulate human non GC DLBCL and can be a useful model mouse for drug discovery targeting tumor microenvironment including immunocheck point drugs. [Display omitted] DisclosuresKotani:Janssen Pharma: Research Funding.

Genetically-Defined Diffuse Large B-Cell Lymphoma Subsets Arise By Distinct Pathogenetic Mechanisms and Predict Outcome

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease that largely arises from antigen-exposed B-cells that transit through the germinal center (GC). DLBCL is further classified into transcriptional subtypes - activated B-cell (ABC) and GC B-cell (GCB). ABC DLBCLs exhibit genetic alterations in NF-kB modifiers and proximal components of the B-cell receptor (BCR) pathway and perturbed terminal B-cell differentiation. GCB DLBCLs have reported alterations in chromatin-modifying enzymes, PI3K signaling and Gα-migration pathway components and frequent translocations of BCL2 .Despite the recognized molecular heterogeneity in DLBCL, previously published genomic analyses have largely focused on single types of alterations - mutations, copy number alterations (CNAs) or structural variants (SVs) - in smaller data sets with more limited clinical annotation. An unbiased comprehensive analysis of all three alteration types is needed to define discrete, clinically annotated subtypes of DLBCL.We performed whole exome sequencing (WES) of 304 newly diagnosed DLBCLs, using an expanded bait set that captures recurrent SVs; 85% of study patients were uniformly treated with R-CHOP and had long-term follow-up. Somatic alterations (mutations, CNAs and SVs) and their clonality were determined with analytical pipelines developed at the Broad Institute. Notably, half of our DLBCL cohort lacked patient-matched normal samples, prompting the successful development of new methods to analyze tumor-only WES data. Significantly mutated genes (SMGs) were identified with MutSig2CV andrecurrent CNAs were defined with GISTIC2.0 .With increased sample size and improved methodology, we identified 158 SMGs, CNAs and SVs; the 98 SMGs included ones previously unreported in DLBCL but described in other lymphoid malignancies or cancer. Additional insights into the putative biological function of newly identified alterations were obtained by overlaying the predicted protein changes onto their 3-dimensional protein structures.We analyzed the mutational signatures in these DLBCLs and found that the majority of exome mutations were caused by spontaneous deamination at CpGs, a clock-like genetic signature associated with aging; only a minority of mutations were attributed to activation-induced cytidine deaminase (AID), an enzyme required for physiologic immunoglobulin receptor editing and aberrant somatic hypermutation.IGH, BCL2, BCL6 and MYC were the most frequently rearranged genes (40%, 21%, 19% and 8%, respectively). There were 18 arm-level and 18 focal regions of copy gain and 2 arm-level and 32 focal regions of copy loss with frequencies ranging between 5-32%.SMGs were significantly more likely to reside within focal CNAs (p=1x10-44), suggesting that these driver genes were perturbed by multiple mechanisms. Individual DLBCLs had a median of 17 genetic drivers, highlighting the need for a more comprehensive analysis. Therefore, we applied an integrated clustering approach to the recurrent mutations, CNAs and SVs and delineated 5 genetically distinct DLBCL subtypes and determined the likely temporal order of alterations within each cluster.This unbiased approach identified a previously unappreciated favorable risk ABC subset with genetic features of an extrafollicular, possibly marginal zone origin; a more tightly defined poor risk group of ABC DLBCLs with frequent BCL2 gain and concordant MYD88L265P / CD79B mutations; a distinct poor risk subset of GCB tumors with BCL2 SVs and alterations of PTEN and epigenetic enzymes and a discrete group of good risk GCB DLBCLs with specific alterations in BCR/PI3K, JAK/STAT and BRAF pathway components and multiple histones; and a Cell-of-Origin-independent subset with biallelic inactivation of TP53, 9p21.3/CDKN2A and associated genomic instability. These findings likely explain the variability in outcome predictions with the binary ABC- vs. GCB-DLBCL transcriptional classification and the challenges of therapeutically targeting less well defined DLBCLs.The genetically distinct DLBCL subsets provide a framework for assessing previously unrecognized heterogeneity in this disease, characterizing combinations of genetic alterations that drive DLBCL biology and guiding the development of rational single-agent and combination therapies in patients with the greatest need. DisclosuresCerhan:Janssen: Other: Scientific Advisory Board (REMICADELYM4001); Janssen: Other: Multiple Myeloma Registry Steering. Rodig:Bristol-Myers Squibb: Honoraria, Research Funding. Neuberg:Synta Pharmaceuticals: Other: Stock shares. Shipp:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Other: Scientific Advisory Board; Cell Signaling: Honoraria; Bayer: Research Funding; Takeda: Other: Scientific Advisory Board; Merck: Other: Scientific Advisory Board; AstraZeneca: Honoraria.

Preclinical Evaluation of eFT226, a Novel, Potent and Selective eIF4A Inhibitor with Anti-Tumor Activity in B-Cell Malignancies

Translational control of oncoprotein expression has been implicated in the pathogenesis of multiple solid tumors and hematological malignancies. Protein synthesis is tightly regulated largely at the initiation stage through the eukaryotic translation initiation factor 4F (eIF4F) complex. eIF4A, an RNA helicase, is an essential component of the translation initiation complex and selectively regulates a subset of mRNAs based on the sequence and structure of the 5‘-untranslated region (UTR). B-cell receptor signaling activates eIF4A resulting in the selective upregulation of oncogenes involved in cell proliferation, survival and metastasis. B-cell malignancies are often associated with dysregulation of oncogenes or anti-apoptotic proteins (e.g., c-MYC, CCND1/3, BCL2 and MCL-1) that contain structured 5‘-UTRs and require enhanced eIF4A activity for translation.eFT226 is a potent and selective eIF4A inhibitor that promotes eIF4A binding to specific mRNA sequences and interferes with the assembly of the eIF4F initiation complex. eFT226 selectively inhibits translation of mRNAs containing longer 5‘-UTRs, an increased frequency of uORFs (upstream open reading frame), and polypurine and/or G-quadraplex recognition motifs. The sequence dependency of eFT226 translational inhibition was evaluated in an in vitro translation assay demonstrating potent inhibition of reporter constructs containing a polypurine motif in the 5‘-UTR (IC50 ~2 nM). Direct binding studies also confirmed the formation of a stable ternary complex between eFT226, eIF4A and mRNA oligonucleotides containing polypurine motifs. This results in selective and translational downregulation of a subset of genes that are important for tumor growth and survival. eFT226 showed potent anti-proliferative activity (EC50 < 15 nM) and an induction of apoptosis against a panel of B-cell lymphoma cell lines. Sensitivity to eFT226 was associated with dose-dependent decreases in the oncogenic drivers c-MYC, CCND1/3, BCL2 or MCL-1 in ABC (activated B-Cell) and GCB (germinal center B-cell) DLBCL (Diffuse Large B-cell Lymphoma) subtypes and Burkitt's lymphoma. eFT226 has good pharmacokinetic properties that result in potent in vivo efficacy with ≤ 1 mg/kg/week IV administration. Preclinical efficacy studies using eFT226 showed in vivo activity across hematological tumor models suggesting that eFT226 may be active in tumor types where overexpression or amplification of oncogenes such as c-MYC, CCND1/3, BCL2 or MCL-1 play a role in disease pathogenesis. These results demonstrate the potential of eFT226 for the treatment of B-cell malignancies. Clinical trials in patients with hematological and other malignancies are planned. DisclosuresThompson:eFFECTOR Therapeutics: Employment, Equity Ownership. Eam:eFFECTOR Therapeutics: Employment, Equity Ownership. Young:eFFECTOR Therapeutics: Employment, Equity Ownership. Fish:eFFECTOR Therapeutics: Employment, Equity Ownership. Chen:eFFECTOR Therapeutics: Employment, Equity Ownership. Barrera:eFFECTOR Therapeutics: Employment, Equity Ownership. Howard:eFFECTOR Therapeutics: Employment, Equity Ownership. Parra:eFFECTOR Therapeutics: Employment, Equity Ownership. Molter:eFFECTOR Therapeutics: Employment. Staunton:eFFECTOR Therapeutics: Employment, Equity Ownership. Hung:eFFECTOR Therapeutics: Employment, Equity Ownership. Parker:eFFECTOR Therapeutics: Employment, Equity Ownership. Chiang:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Wegerski:eFFECTOR Therapeutics: Employment, Equity Ownership. Nevarez:eFFECTOR Therapeutics: Employment, Equity Ownership. Clarine:eFFECTOR Therapeutics: Employment, Equity Ownership. Sperry:eFFECTOR THERAPEUTICS: Employment, Equity Ownership. Xiang:eFFECTOR Therapeutics: Employment, Equity Ownership. Tran:eFFECTOR Therapeutics: Employment, Equity Ownership. Nilewski:eFFECTOR Therapeutics: Employment, Equity Ownership. Packard:eFFECTOR Therapeutics: Employment, Equity Ownership. Michels:eFFECTOR Therapeutics: Employment, Equity Ownership. Sprengeler:eFFECTOR Therapeutics: Employment, Equity Ownership. Ernst:eFFECTOR Therapeutics: Employment, Equity Ownership. Reich:eFFECTOR Therapeutics: Employment, Equity Ownership. Webster:eFFECTOR THERAPEUTICS: Employment, Equity Ownership.