Germinal Center B-cell Research Articles

Real-World Outcomes in Patients with Transformed Follicular Lymphoma Treated with CD19-Targeting CAR-T Therapy

INTRODUCTION. CD19-targeting CAR-T cell therapy has changed the treatment landscape of refractory/relapsed large B cell lymphoma, including transformed from FL (tFL). Axicabtagene ciloleucel (axi-cel), lisocabtagene maraleucel (liso-cel), and tisagenlecleucel (tisa-cel) are now FDA-approved for the treatment of tFL, but limited data are available on their clinical activity in patients (pts) with tFL since outcomes in those pts were not separately reported in registrational studies. Here, we report a single center's experience with CD19-directed CAR-T cell therapy in pts with tFL. METHODS. We performed a retrospective analysis of 54 pts with tFL treated with CAR-T cell therapy from January 2018 to December 2022. Pathological evidence of histologic transformation (HT) was confirmed. The main clinical-biological characteristics of the pts were extracted from the electronic medical records. Cytokine release syndrome (CRS) and neurotoxicity were graded according to ASTCT guidelines. Progression-free (PFS) and overall survival (OS) were measured from the time of CAR-T cell infusion; PFS events were death, relapse, and disease progression. RESULTS. Within our cohort of patients with tFL, the median age was 67 years (range 42-81), 65% were male and 77% had stage III-IV disease. Eight of the pts (15%) had an ECOG Performance Status (ECOG-PS) ≥2. Median time to transformation was 27 months from original FL diagnosis (range 0 to 223), including 6 cases with composite lymphoma (FL + aggressive histology) and 4 with synchronous transformation. Twenty percent presented with high-grade B-cell lymphoma (HGBCL) with MYC and BCL2 and/or BCL6 rearrangements. Using Hans' algorithm, 85% (44/52) were identified as a germinal center B-cell origin (GCB). In 22 cases, targeted sequencing using the MSK-IMPACT panel was performed at the time of HT or prior to CAR-T. TP53 mutation/deletion was present in 50% of the cases (Figure 1). Pts received a median of 3 (range 1-9) prior lines of therapy including those for FL, and 9 pts (17%) had undergone prior autologous stem cell transplantation (SCT). Thirty-four pts (63%) were refractory to the last therapy, including 29 pts (53.7%) with primary refractory disease after HT. Forty-one pts (76%) received bridging therapy. Seventy-two percent of pts received axi-cel, 19% tisa-cel, and 9% liso-cel. CRS of any grade was seen in 43 (80%) pts with grade >3 in 3 (6%). Neurologic toxicity of any grade was observed in 20 (37%) pts with grade >3 in 7 (13%). The median duration of follow-up was 26.2 months. The overall response rate was 87%, including 67% complete response (CR). The median response duration (DOR) was 11 months, with a 2-year DOR of 42% (95% CI: 28-61%). Objective responses were achieved in pts with high-risk features such as HGBCL, TP53 mutation, and chemotherapy-refractory disease. Twenty-nine pts experienced relapse/progression during follow-up, with a median PFS for the entire cohort of 9.8 months and a 2-year PFS of 40% (95% CI: 28.3-56.3%) (Figure 2). Refractory disease after HT and high LDH serum levels were associated with a significantly lower PFS (median PFS not reached vs. 6.4 months, P=0.031; 24.4 vs. 3.2 months, P=0.003 respectively). Notably, all pts who achieved PR eventually progressed. In 23 pts with relapse, a biopsy was obtained, showing DLBCL or HGBCL morphology in all instances. Interestingly, all relapses occurred during the first 24 months. Twenty-six pts received salvage therapy after CAR-T progression, with a median of 1 line (range 1-7). Out of these, eight (31%) pts achieved a CR and went to allogenic SCT as a consolidative strategy. Twenty-two pts died during follow-up, 19 due to progression and 3 of non-lymphoma causes, including 2 of COVID-19 infection. The median OS was 31 months, and the 2-year OS rate was 57% (95% CI: 44.3-73.8%) (Figure 2). Median OS was not reached among the 36 pts who achieved a complete response. CONCLUSIONS. CD19-targeting CAR-T cell therapy is effective for pts with relapsed/refractory tFL, including patients with characteristics of poor prognosis, with efficacy and toxicity profiles comparable to that observed in previous DLBCL studies. Unfortunately, pts who relapse post-CAR-T therapy have limited effective therapeutic options available, and this currently remains an unmet medical need within the field.

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.

Development of Syngeneic Murine Cell Lines of Germinal Center-Derived B-Cell Lymphomas

Background. Germinal center (GC)-derived B-cell lymphomas are clinically and biologically heterogeneous. Next generation sequencing has enabled identification of genetic aberrations involved in lymphoma pathogenesis, which may provide prognostic predictions and potential therapeutic targets. However, the mechanisms driving pathogenesis are still not fully understood, especially in the context of an intact tumor microenvironment, in part because there are no available syngeneic models of lymphoma faithfully representing the pathological features of human disease. Therefore, there is a critical need for lymphoma models which can be studied in immunocompetent mice. Methods. We established eight murine cell lines derived from spontaneous B-cell lymphomas with GC origin arising in genetically engineered mouse models (GEMM). These cell lines were developed by passing an original tumor from a GEMM through several passages of Rag1-/- and wild type mice, which enabled selection of more fit/aggressive clones that grow in vitro. We also tested multiple culture conditions and characterized the lines via flow cytometry, VDJ and targeted sequencing, karyotyping, and evaluation of tumor development and its microenvironment in vivo. Results. This resource describes eight cell lines driven by recurrent genetic mutations found in diffuse large B-cell lymphoma and follicular lymphoma patients. The first cell line is defined by (1) BCL2 overexpression (hereafter B2). Four cell lines couple BCL2 overexpression with epigenetic dysregulation, defined by (2) BCL2 and gain-of-function (GOF) mutation of EZH2 Y641F (hereafter EZB), (3) EZB and MYC overexpression (hereafter EBM), (4) BCL2 with knockout of KMT2D (hereafter KB), and (5) BCL2 with knockout of SETD2 (hereafter S2B). Finally, three cell lines are defined by mutations to the Toll-like and B-cell receptors pathways, defined by (6) BCL2 and GOF mutations in Myd88 L252P and CD79b Y195H (hereafter MBC79), (7) BCL2, Myd88 L252P and GOF mutation in Card11 L251P (hereafter MBC11), and (8) BCL2 and Card11 L251P (hereafter BC11). Doubling time of these lines ranges from 30-72 hours, with EZB and EBM on the fast end and S2B on the slow end. By flow cytometry, we show that B2, MBC79, and MBC11 lose B-cell surface markers and are very plasmacytic (>95% B220- CD19- CD138+). In contrast, the EZB, EBM, KB, BC11, and S2B are mostly characterized by GC B-cell markers (B220+ CD19+ CD138- FAS+ CD38- GL7+). By VDJ sequencing, we found that EZB, BC11, and S2B are very monoclonal, with one rearrangement arising from the original tumor and taking over to represent >95% of clones. In contrast, the MCB11 and MBC79 lines are very polyclonal, with the top 10 rearrangements being equally represented, and tending to shift between passages. Thus far, S2B and EZB have been shown to grow in immunocompetent mice, representing novel syngeneic lymphoma models. While mice injected with S2B can survive for up to 30 days, mice injected with EZB die significantly earlier. We established two EZB lines from early (P1) and late (P6) passages of a tumor arising from the same original mouse. Notably, we show that mice injected with P1 survive 15-20 days, whereas mice injected with P6 survive 10-15 days. Although both P1 and P6 grow at similar rates in vitro, P1 develops high grade follicular lymphomas in C57BL6 recipients, whereas P6 develops DLBCL, suggesting additional passaging drives selection for more aggressive disease. Imaging of the tumors reveals a lack of follicular pattern. Notably, P6 tumors display less infiltration of CD4 and CD8 T-cells and low abundance or absence of FDC meshwork, as compared to P1. Imaging of mice engrafted with luciferase transduced P1 and P6 shows dissemination into spleen, lymph nodes, liver, and kidney, with more advanced disease in P6 injected mice. Conclusion. While conventional preclinical tumor mouse models are often used for drug sensitivity and treatment prediction studies, they are time-consuming and expensive. These cell lines serve as a preclinical tool to test novel therapeutic strategies and develop a deeper understanding of the molecular mechanisms driving lymphomagenesis, therapeutic response, and resistance in genetically defined subtypes of GC-derived B-cell lymphomas. Most importantly, this resource meets a critical need in the field for syngeneic models of lymphoma to study disease progression and precision therapy in immunocompetent mice.

SWI/SNF Chromatin Remodeling Complex Orchestrates Sequential Binding of Key Transcription Factors in B Cells and Restricts Aggressive Lymphoma

Mutations in the SWI/SNF chromatin remodeling complex BAF are a recurring feature in many cancers, with a high prevalence in follicular lymphoma (FL; >19%) and diffuse large B-cell lymphoma (DLBCL; >34%). Despite this, the mechanism that links mutations in ARID1A, most frequently mutated BAF subunit, to the development of lymphoma is still not understood. To this aim, we crossed Arid1a-floxed mice with Cγ1-Cre mice to yield offspring with a conditional (cKO) Arid1a deletion in germinal center (GC) B cells, FL and DLBCL cell-of-origin. We immunized WT/WT ( Arid1a+/+; Cγ1Cre/+) and cKO/WT ( Arid1a+/-; Cγ1Cre/+) mice with sheep red blood cells and analyzed 10 days later at the peak of the GC reaction. Upon Arid1a deletion, we found a decrease in the total number of GC B cells relative to total B cells (WT/WT vs. cKO/WT; p<0.001). However, we observed skewing of GC polarity manifesting as increased proportions of proliferating centroblasts (CB) vs. centrocytes (CC) (WT/WT vs. cKO/WT; p=0.004). These observations imply a substantial dysfunction at the CC stage, which could account for the overall reduced number of GC B-cells. To investigate this, we performed a detailed molecular characterization of these cells. We performed the ATAC-seq assay on sorted CB and CC from our mouse model and observed an extensive loss of chromatin accessibility. Changes in chromatin accessibility are thought to be dependent on the recruitment of the BAF complex by specific transcription factors. We discovered strong enrichment for DNA motifs of PU.1 and NF-kB factors in closing chromatin (p-value <0.001), indicating their dependence on the BAF complex. Furthermore, we noted that this chromatin closing occurred in proximity to canonical GC exit programs, including genes induced by the CD40, NF-kB signaling, IRF4, STAT3, IL2, IL4, IL6 and Notch pathways (hypergeometric p-value <0.001), indicating GC-exit perturbation. Expanding upon our findings, we leveraged a combined single-cell RNA and ATAC assay (Multiome) to scrutinize subtle shifts within GC populations. Determining the chromatin accessibility within the pseudo-time trajectory of GC transitions, we found that PU.1 and NF-kB factors are chronologically dependent on ARID1A with PU.1 accessibility initially compromised, followed by a subsequent decrease at binding sites for NF-kB factors. This sequence reveals ARID1A's unrecognized function as a regulator of temporal dynamics of these key transcription factors. Strikingly, our Multiome data revealed expansion of the pre-memory B cells accompanied by a decrease in the proportion of pre-plasma cells upon Arid1a deletion (p-value <0.001), suggesting that Arid1a loss favors GC exit towards the memory cell fate. To validate our findings, we performed immunophenotyping and observed an increase in memory B cells in cKO/WT mice (p=0.019), and a decrease in long-lived plasma cells (p=0.012) upon NP-KLH immunization. Furthermore, we observed that the absence of ARID1A tilts GC cell-fate towards immature IgM+CD80-PDL2- memory B cells (p=0.0016), known for their potential to re-enter new GCs. Linking this critical role of ARID1A in chromatin regulation to lymphomagenesis, we observed a reduction in overall survival for mice carrying VavP-Bcl2; Arid1a+/- allele (compared to VavP-Bcl2; Arid1a+/+; p=0.0087). Remarkably, we further show that FL patients with ARID1A-inactivating mutations display an immature memory B-cell-like state with increased transformation risk to aggressive disease (p=0.0391). These observations offer mechanistic understanding into the emergence of both indolent and aggressive lymphomas in ARID1A-mutant patients through formation of immature memory-like clonal precursors.

Optogenetic Control of Oncogenic Signaling in B-Cell Malignancies

Background: B-cell receptor (BCR) signals are essential determinants of survival and proliferation throughout normal B-cell development. In B-cell malignancies, these signals are frequently generated by oncogenic mimics of the BCR signaling pathway. For instance, oncogenes in B-ALL, derived from B-cell precursors, typically mimic survival signals from a constitutively active pre-BCR, while tonic and chronic active BCR signaling were identified in mature B-cell lymphomas. Oncogenic BCR-signaling represents an important target of therapeutic intervention: for instance, small molecule inhibitors of SYK (e.g. entospletinib) and BTK (e.g. ibrutinib) have been developed to disrupt oncogenic BCR signaling in mature B-cell lymphomas. Besides SYK and BTK tyrosine kinases, oncogenic BCR-signaling leads to activation of PLCG2, which initiates the release of Ca 2+ from the ER into the cytoplasm. Thereby, calcium flux is decoded by NFATC1 or NF-kB based on fast (NFATC1) or slow (NF-kB) frequencies of Ca 2+-signals. Significance: To interrogate Ca 2+-signals as critical integration point of oncogenic BCR-signaling, we engineered cell lines and PDX with a GCaMP6s-biosensor, which allows tracing of calcium signaling in single cells over time ( Figure A). To cover B-cell malignancies from multiple stages of B-cell development, we engineered B-ALL (pro- and pre-B cells), mantle cell lymphoma (MCL, naïve B-cells), Burkitt's lymphoma (germinal center), DLBCL (post-GC), multiple myeloma (terminally differentiated plasma cells) and Hodgkin's disease (“crippled” BCR-deficient GC-B cells). Strikingly, B-cell malignancies exhibit autonomous Ca 2+-oscillations, which decreased in their frequency from 20 mHz (pro- and pre-B), 11 mHz (naïve), 4 mHz (germinal center) to 0 mHz in post-GC and terminally differentiated plasma cells based on the differentiation stage of their cell of origin ( Figure A). Results: Given the striking differences in oncogenic BCR-signaling as measured by autonomous Ca 2+ oscillations, we developed an optogenetic system to control the frequency and amplitude of Ca 2+ oscillations by blue light pulses ( Figure B). To model frequency-modulated Ca 2+ signaling in B-cells, we engineered murine pre-B cells carrying an optogenetic tool termed OptoCRAC, which enables reversible activation and inactivation of the plasma membrane Ca 2+ channel Orai1 by intermittent blue light irradiation. Time-lapse imaging of a fluorescent Ca 2+ reporter (R-CaMP1.07) confirmed rapid and reversible Ca 2+ influx in response to intermittent blue light pulses. To assess the phenotypic consequence of high-frequency Ca 2+ oscillations, we developed an LED-array-based optogenetic platform for cell culture plates. While low-frequency Ca 2+ oscillations (0.5 mHz) did not impact cell viability, high-frequency Ca 2+ oscillations (20 mHz) rapidly induced NFAT activation (5 min), and eventually cell death within 72 h ( Figure B). Mechanism and conclusions: To elucidate the mechanistic contribution of NFATC1 in B-cell death induced by fast Ca 2+ oscillations, we tested whether genetic deletion of Nfatc1 is sufficient to rescue B-cell death upon fast Ca 2+ oscillation induced by 20 mHz intermittent blue light pulses. As expected, while B-ALL cells retaining intact Nfatc1 rapidly decreased cell viability, the cells with Cre-mediated deletion persistently remained in cell culture upon 20 mHz Ca 2+ oscillation. In addition to genetic deletion of Nfatc1, we also found that inducible expression of mutant activators of NF-kB signaling were able to rescue B-cells from cell death induced by high-frequency oscillations: Concurrent expression of Card11 L232LI, MYD88 L265P and IKK2 S177E/S181E not only reduced the frequency of autonomous Ca 2+ oscillations but also enabled B-cell survival despite delivery of light-pulses and Ca 2+ oscillations at a fast 20 mHz frequency. We conclude that autonomous Ca 2+ oscillations represent a critical integration point of oncogenic BCR-signaling in B-cell malignancies and that optogenetic control of BCR-downstream Ca 2+ oscillations will reveal previously unrecognized vulnerabilities that can be exploited to increase efficacy of BCR-signaling inhibitors (e.g. ibrutinib, entospletinib, idelalisib) that are currently being used for the treatment of B-cell lymphomas.

The genetic landscape of histologically transformed marginal zone lymphomas.

Marginal zone lymphomas (MZLs) comprise a diverse group of indolent lymphoproliferative disorders; however, some patients develop histologic transformation (HT) with rapid progression to aggressive lymphoma. Forty-three MZLs with HT (HT-MZLs), 535 MZLs, and 174 de novo diffuse large B-cell lymphomas (DLBCLs) without rearrangements of MYC, BCL2, and BCL6 were collected. Among these, 22 HT-MZLs, 39 MZLs, and 174 DLBCLs were subjected to 148-gene targeted exome sequencing. The clinicopathologic features of patients who had HT-MZL and their genetic alterations were compared with those of patients who had MZLs and DLBCLs. All 43 HT-MZLs corresponded to DLBCLs. No HT-MZLs harbored BCL2 and MYC and/or BCL6 rearrangements. Bone marrow involvement and higher levels of lactate dehydrogenase were significantly more common in HT-MZLs than in MZLs. Furthermore, upregulated BCL6, MUM1, C-MYC, and Ki-67 expression was observed more frequently in HT-MZLs than in MZLs. TBL1XR1 was the most frequently altered gene (63.6%) in HT-MZLs, followed by CCND3 (31.8%), CARD11, ID3, and TP53 (22.7%). A trend toward worse progression-free survival in patients with TBL1XR1 mutations was observed. Compared with MZLs and non-germinal center B-cell (GCB) type DLBCLs, significantly higher frequencies of TBL1XR1 and ID3 mutations were identified in HT-MZLs. PIM1 mutations frequently occurred in DLBCLs and were significantly associated with TBL1XR1 mutations but were mutated less in HT-MZLs that had TBL1XR1 mutations. The current findings reveal the clinicopathologic and genetic features of HT-MZLs, suggesting that these tumors might constitute a group distinct from MZL and de novo non-GCB type DLBCL. TBL1XR1 mutations may be considered a predictor of HT in MZL.

T-Cell Signaling Mediates the Epigenetic Priming of Germinal Center B-Cell Plasticity and Stem Functionality

Chemo-immunotherapy can prolong life expectancy of patients with Diffuse Large B-cell Lymphomas (DLBCL). However, the emergence of donor-derived lymphoma cases after stem cell transplantation and the relapse of a third of DLBCL patients, strongly suggests the existence of persisting lymphoma repopulating cells (LRC) with stem-like functionality. This apparently controversial concept arises from the fact that DLBCLs originate from mature rather than progenitor cells. Identifying the biological and epigenetic features of these elusive LRC, and how they arise from normal germinal center (GC) B cells, is therefore critical and remains underexplored. Although fully mature, GC B cells naturally manifest remarkable phenotypic plasticity as compared to other cell types. We hypothesized that this plasticity reflects stem-like programming and functionality. Along these lines, we found that GC B cells are uniquely enriched for pluripotent stem cell transcriptional signatures, and increased chromatin accessibility at stem cell enhancers. We reasoned that the capacity to undergo induced Pluripotent Stem Cells (iPSC) reprogramming could serve as a surrogate assay for stem-like plasticity. Therefore, we investigated the potential of GC B cells to form iPSCs using a doxycycline-dependent mouse strain that enables the inducible expression of Yamanaka transcription factors (TF) in any given cell. Strikingly, GC B cells isolated from our mice manifested a 10-fold (p<0.0001) increase in iPSC reprogramming as compared to other mature B cells. GC B cells are a heterogenous mix of functionally distinct subpopulations. Using single cell RNA-seq and iPSC assays in sorted cells, we noted that the rare population (~3%) of GC B cells selected by T-cell help manifested the stem-like transcriptional profile and iPSC reprogramming phenotype. Moreover, blocking T-cell help during the GC reaction in vivo using CD40 blockade abrogated iPSC forming capacity. Lymphoma-associated EZH2 Y641F mutations impair GC B-cell interactions with T FH cells due to aberrant repression of immune synapse genes. Accordingly, EZH2 Y641F GC B cells manifested loss of the iPSC phenotype. T-cell help induces MYC in GC B cells. However, induction of MYC in mature naïve B cells did not confer them with iPSC potential indicating that other mechanisms must be at play. Lymphoma-associated Btg1 Q36Hmutations confer fitness in GC B cells by enhancing their response to T-cell help. Reciprocal to EZH2 Y641F, Btg1 Q36Hmutant GC B cells manifested further significant increase in iPSC formation. Performing multi-ome (simultaneous single-cell RNAseq and ATACseq) studies in GC B cells revealed massive gain in chromatin accessibility of bona fide stem cell super-enhancers in GC B cells that received T-cell help. There was also a reduced accessibility at genes that maintain B-cell lineage (e.g. Pax5) and GC phenotypes (e.g. Foxo1). Finally, lymphoma-associated Histone 1 mutations induce upregulation of stem cell programs in GCs and could enhance iPSC reprogramming in fibroblasts. We therefore hypothesized that H1 deficiency would overcome the GC dependency on T-cells for their plasticity phenotype. Indeed, increased iPSC formation occurred across all GC subpopulations in H1 C/E-/- (HIST1C/HIST1E knockout) mice. We speculate that restricting plasticity to B cells under selection by T cells, limits the potential for GC B cells to acquire high levels of plasticity, hence reducing the potential for these cells to initiate lymphomas. Consistent with this notion, we found that DLBCL patients enriched for these stem cell signatures manifested inferior clinical outcomes.

Impact of “Cell-of-Origin” on Outcome after Axicabtagene-Ciloleucel CAR-T Cell Therapy in Relapsed/Refractory Aggressive Non-Hodgkin B-Cell Lymphomas

Introduction: Despite significant advances in characterizing aggressive B-cell non-Hodgkin lymphomas (aNHL) based on molecular and genetic signatures, the Hans algorithm, determined by immunohistochemistry, remains the most commonly used approach to differentiate aNHLs based on the cell-of-origin (COO) into germinal center B-cell (GCB) and non-GCB types. Although the Hans algorithm has demonstrated reliability as a predictor of better outcomes in newly diagnosed GCB cases, conflicting evidence has emerged, particularly in the relapsed/refractory (R/R) setting, and there are no definitive data about the impact of COO in patients (pts) receiving CAR-T cells. Thus, we conducted a retrospective analysis to evaluate the impact of COO extended to R/R aNHLs who underwent treatment with axicabtagene-ciloleucel (axi-cel). Methods: We retrospectively collected data from pts with R/R aNHLs treated with axi-cel at Fred Hutchinson Cancer Center between February 2018 and August 2022. The Hans algorithm, using CD10, BCL6 and MUM1 markers, was employed to stratify the pts into two cohorts: GCB and non-GCB. Paired biopsies of diagnosis and relapse before CAR-T eligibility were reviewed at our institution. Response to CAR-T was assessed at one month per Lugano PET/CT criteria. Univariate cox proportional hazard regression was used to estimate associations between baseline factors and outcomes. Statistically significant factors ( p<.05) in univariate analysis were further evaluated by multivariate analysis. Continuous variables were compared by Mann-Whitney U test and categorical variables by Fischer's exact test. Progression-free survival (PFS) was calculated from the date of CAR-T cell infusion to the date of death from any cause, disease progression or relapse. Overall survival (OS) was calculated from the date of CAR-T cell infusion to the date of death from any cause. Results: Sixty-four pts were included in this analysis, with an equal distribution of pts in the two cohorts: 32 with GCB subtype and 32 with non-GCB subtype. Baseline pts' characteristics and histology subtypes are listed in Table 1. Transformed follicular lymphoma and primary mediastinal B-cell lymphoma were not included in this analysis. The presence of bulky disease (defined as >5cm diameter) was higher in the GCB subtype (56% vs 22%, p<.001). Although primary refractory cases were more frequent in the GCB cohort (87.5 vs 66%), statistical significance was not reached (NR)( p=.07). The same proportion of pts received bridging therapy in the two groups (53% vs 53%, p=.99). Pts evaluable for response assessment were 62. Median time to best response was 29 days (range, 21-186) for GCB pts and 31 days (range, 22-182) for non-GCB pts ( p=.91). The overall response rate was 70% for GCBs (34.5% complete response [CR], 34.5% partial response [PR]) and 75% for non-GCBs (44% CR, 31% PR). After a median follow-up of 24.5 months (range, 9-121), comparable outcomes were observed for both the COO groups: despite the estimated median OS of 14 months (95% CI, 4.73-NR) for GCB vs 40 months (95% CI, 12.55-67.46) for non-GCB, we did not find any significant differences ( p=.73) (Figure 1A). Estimated median PFS was 10 months (95% confidence interval [CI], 3.5-16) for GCB and 11 months (95% CI, 0-24) for non-GCB ( p=.57) (Figure 1B). The 2-year OS and PFS were: GCB, 45% (95% CI, 30-69) and 44% (95% CI, 29-67) respectively; non-GCB, 55% (95% CI, 38-79) and 29% (95% CI, 16-55), respectively. Treatment-related toxicities did not differ between GCB and non-GCB, with cytokine-release syndrome rates of 97% vs 94% ( p=.99) and neurotoxicity rates of 53% vs 62.5% ( p=.61), respectively. The incidence of infectious complications was 25% vs 19% ( p=.76). On univariate analysis, the impact of COO on OS (HR=0.82, 95% CI 0.39-1.73, p=.60) and PFS (HR=1.18, 95% CI 0.61-2.28, p=.62) was undetermined. After adjusting for bulky disease, elevated LDH, absolute lymphocyte count≤1000/uL, performance status≥2, >2 lines of therapy and response status before axi-cel infusion, the impact of COO on OS (adjusted HR [aHR]=1.73, 95% CI 0.71-4.22, p=.23) and PFS (aHR=2.01, 95% CI 0.93-4.35, p=.08), remained undetermined. Conclusion: We could not confirm an association between COO, as determined by Hans algorithm, and outcomes in R/R aNHLs who received axi-cel at our center. Prospective analyses on larger datasets and other types of CAR-T products are needed.

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.

Glofitamab Plus R-CHOP Induces High Response Rates with a Manageable Safety Profile in Patients with Previously Untreated Diffuse Large B-Cell Lymphoma (DLBCL): A 12-Month Analysis from a Phase Ib Study

Background: Over a third of patients (pts) with DLBCL do not respond or relapse after first-line (1L) treatment with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP; Sarkozy and Sehn. Ann Lymphoma 2019); these pts have limited curative options. Glofitamab (Glofit) is a T-cell engaging bispecific antibody with a novel 2:1 (CD20:CD3) format that allows bivalent binding to CD20 on B cells; this enables combination with anti-CD20 antibodies, including rituximab. Glofit monotherapy has shown durable responses in pts with relapsed/refractory DLBCL (Dickinson et al. NEJM 2022). In the safety run-in and expansion phases of the ongoing NP40126 trial (NCT03467373), Glofit + R-CHOP demonstrated promising efficacy and manageable safety in pts with 1L DLBCL (Topp et al. ASH 2022). Here, we report durability of response beyond 12 months in Glofit + R-CHOP-treated pts with 1L DLBCL from the NP40126 trial. Methods: Pts received R-CHOP for 6-8 three-weekly cycles. Intravenous Glofit was given with step-up dosing during Cycle (C)2 (Day [D]8, 2.5mg; D15, 10mg) and at the target dose (30mg) from C3D8 onwards. Glofit maintenance was allowed for up to one year. Efficacy was analyzed in all pts who had been on the study long enough to have a protocol scheduled end-of-treatment response assessment. Safety was analyzed in all pts who received at least one dose of any study drug. Response was assessed by PET-CT using Lugano criteria (Cheson et al. JCO 2014). Cytokine release syndrome (CRS) events were graded using the American Society for Transplantation and Cellular Therapy criteria (Lee et al. Biol Blood Marrow Transplant 2019) and other adverse events (AEs) by the Common Terminology Criteria for Adverse Events (v4.0). Hospitalization was not mandated for Glofit. Results: As of April 4, 2023, 56 pts were enrolled in this cohort. Median age was 68 years (range: 21-84), 54 pts (96.4%) had Ann Arbor stage III/IV disease, and median International Prognostic Index (IPI) score was 3 (IPI 1: 3.6% [2/56], IPI 2: 33.9% [19/56], IPI 3: 35.7% [20/56], IPI 4: 23.2% [13/56], IPI 5: 3.6% [2/56]). The cell of origin was germinal center B-cell (GCB) in 25 (44.6%) pts and non-GCB in 11 (19.6%) pts. After 17.1 (range: 0-26) months of median follow-up, the best overall response rate was 92.9% (52/56) and the complete metabolic response (CMR) rate was 83.9% (47/56). Median time to CMR was 1.8 months (95% confidence interval [CI]: 1.6-4.7). Most CMRs (44/47; 93.6%) were ongoing at data cut-off ( Figure). Among complete responders, the Kaplan-Meier estimated probability of ongoing responses at 12 months was 91.5% (95% CI: 82.2-100). Median duration of response and duration of complete response were not reached at data cut-off. The incidence of AEs and serious AEs (SAEs) were similar compared with earlier analyses. Grade (Gr) ≥3 AEs occurred in 42 (75.0%) pts and Gr ≥3 AEs related to Glofit in 14 (25.0%) pts. SAEs were reported in 20 (35.7%) pts and SAEs related to Glofit in six (10.7%) pts. Gr 5 AEs were reported in four (7.1%) pts (COVID-19 pneumonia, n=3; infusion related reaction associated with rituximab, n=1). AEs leading to dose interruption of Glofit occurred in 12 (21.4%) pts. Median dose intensity was 100% for all R-CHOP components. AEs leading to discontinuation of Glofit occurred in one (1.8%) patient. CRS events were reported in six (10.7%) pts: Gr 1, n=4; Gr 2, n=2; no Gr 3/4 CRS events were reported. All CRS events occurred during C2-3. Tocilizumab was used in two (33.3%) pts with CRS. Neurologic AEs (NAEs) occurred in 23 (41.1%) pts (most common NAEs (>5%) were peripheral neuropathy, n=7 [12.5%]; paraesthesia, n=5 [8.9%]; insomnia, n=4 [7.1%]; headache, n=3 [5.4%]); Gr 3 NAEs included cerebrovascular accident (n=1) and herpes zoster (n=1). No Glofit-related NAEs consistent with immune effector cell-associated neurotoxicity syndrome were reported. Neutropenia was reported in 26 (46.4%) pts (Gr ≥3 neutropenia: Gr 3, n=5; Gr 4, n=19). Infections and infestations were reported in 27 (48.2%) pts (of which the majority were Gr ≤2 [17/27]); Gr 3, n=7; Gr 5, n=3. Conclusions: Glofit + R-CHOP had a manageable safety profile with a low incidence of CRS, and continued to demonstrate durable responses with an improved CMR rate since the last data cut-off. These findings support the potential for favorable long-term outcomes with Glofit + R-CHOP as 1L treatment of DLBCL. Further analyses, including circulating tumor DNA data, will be presented.

High Frequency Mutant Genes and Prognosis Value of HIV-Related Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) is the most common subtype in AIDS-related lymphoma (ARL) in HIV-infected patients, and the probability of DLBCL in HIV-infected patients is up to 17 times higher than that of the healthy population. However, the genetic mutation profile of HIV-associated DLBCL is still controversial, and the gene regulatory mechanism of HIV infection leading to the high incidence of DLBCL is still unclear. Methods: A total of 12 HIV-positiveDLBCL and 39 HIV-negativeDLBCL patients who treated in Chongqing University Cancer Hospital from March 2022 to October 2022 were enrolled. Tumor tissue samples were selected to detect by second-generation sequencing technology, and gene heat mapping was used to analyze the high-frequency mutant genes in HIV-positive lymphoma patients, comparing the differences mutant genes between HIV-positive and HIV-negative DLBCL patients. The specific mutant genes in HIV-positive DLBCL were screened, and Kaplan-Meier estimate the impact of specific gene changes on disease prognosis in analyze DLBCL lymphoma. Results Compared with HIV-negative DLBCL patients, 12 patients with HIV-positive DLBCL were mostly male (91.7%), and the disease subtype was mostly germinal center B-cell like(GCB) subtype (75%). HIV-positive DLBCL patients were associated with EBV infection, elevated LDH, increased proportion of CD4 + cells < 200 (cell/ul), and more aggressive. The gene heat mapping results showed that TP53, TBL1XR1 and KMT2D had higher mutations in HIV-positive DLBCL and HIV-negative DLBCL patients. Compared with HIV-positive DLBCL, HIV-negative DLBCL patients were more likely to have gene mutations such as IGLL5, KMT2C, CD79B, BTG1, HIST1H1E, TNFRSF14, B2M. Compared with HIV-negative DLBCL patients, HIV-positive DLBCL patients detected more complex gene mutation sites, and were more common to have gene mutations including TP53, LRP1B, MYC, NOTCH2, SPEN, EP300, SOCS1, TYK2, ARID1A, GNA13, MAP2K1. Among those gene mutations, LRP1B, MYC, TYK2 have a higher probability in HIV-positive DLBCL patients (P<0.05). Compared with HIV-negative DLBCL patients, HIV-positive DLBCL patients have a worse prognosis by Kaplan-Meier analysis, and patients with LRP1B, MYC, TYK2 mutations had a longer time to achieve complete remission. Conclusion HIV-positive DLBCL patients are more prone to gene mutations, and the mutant genes are more complex and diverse. LRP1B, MYC and TYK2 gene mutations may play an important role in the occurrence and development of HIV-positive DLBCL and affect the prognosis of patients. Keyword HIV; lymphoma; genetic mutation; prognosis

Detection of Occurrent Somatic Hypermutation and Associated Gene Expression Profile in Single B Cells

Introduction Somatic Hypermutation (SHM) is an endogenous mutator mechanism in B lymphocytes that preferentially targets the immunoglobulin V(D)J coding region and facilitates antibody affinity maturation. The exact frequency and impact of SHM in individual B cells remains elusive, as previous quantifications only considered cumulative outcomes at population level. We therefore employed single-cell RNA sequencing on primary follicular lymphoma (FL) cells, a B-cell malignancy retaining the germinal center (GC) B-cell phenotype, to identify and quantify individual SHM events. This approach allowed us to detect neovariants, defined as single nucleotide differences that originate from recent SHM events, and to explore the gene expression profiles of the B lymphoma cells with neovariants, particularly focusing on genes and pathways effectuating SHM. Methods We collected and processed samples from 12 follicular lymphoma (FL) and five chronic lymphocytic leukemia (CLL) patients as non-germinal center controls. Single cell whole cDNA libraries were prepared by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with semi-nested amplification using 3′ constant domain primers followed by 10X Genomics prep. Both single cell libraries were sequenced in paired-end mode on Illumina HiSeq 4000. We developed a customized method to preprocess scBCR data designed specifically for highly mutated FL BCR sequences. To ensure data quality and validity of putative neovariants, we applied a comprehensive filtering system, including stringent thresholds in terms of numbers of individual immunoglobulin (IG) mRNA molecules sequenced as defined by their UMI, and reads per position obtained from the median of the estimator's distribution, base quality scores, exclusion of cell doublets, position within the V(D)J region, and proportion of cell-level variants. Based on a reported IG mRNA half-life of 3 h and assuming a first-order decay, the filtering criteria implied that detected neovariants were created between 1 and 7 hours prior to the time of sampling. Results A median of 1112 cells (range: 215-16432) per case were analyzed. 1239 IG neovariants were detected in a total of 334 FL cells in 7 of the 12 FL cases. Neovariants were undetectable in control CLL cells. A remarkably high proportion (95,4%) of neovariants were found within known AID motifs (WRCY, WA, and RCG), suggesting active AID involvement in the hypermutation process. The observed fractions of FL cells carrying recent SHM events indicated initiation of FL pathophysiology at least 11,2 months prior to sampling. To further evaluate the role of AID, we compared gene expression profiles between FL cells with and without neovariants. The results demonstrated not only higher expression of AID but as well of pathways involved in SHM. Mismatch repair (hsa03430, 9 enriched genes with p.adjust < 0.001) and base excision repair (hsa03410, 17 enriched genes with p.adjust = 0.01), together with DNA replication (hsa03030, 14 enriched genes with p.adjust < 0.001), were the three most prominent pathways with upregulated genes. In cells with detectable neovariants, the MLH1/MSH2 genes, associated with the recognition of the deamination process, as well as POLH gene, which is involved in error-prone mutations in MMR, were highly expressed. Overall, we observed a more prominent expression of genes related to MMR than BER in cells with neovariants supported by a higher normalized enrichment score (NES) in MMR (MMR:1.93 and BER:1.6). This suggests not only an active SHM process but also potential interplay between AID and MMR in driving SHM in FL. Conclusions Here we report for the first time quantitative detection of ongoing somatic hypermutation at single cell level in FL. Based on the lifespan of IG mRNA, simultaneous detection of two IG mRNA transcripts differing by one (or a few) nucleotides only within a single cell permits to estimate the timing when this SHM event occurred. The detection of this phenomenon in 58% of FL samples and its association with AID expression suggests that AID-induced mutagenesis might be acting at a much higher rate than expected. Quantification of SHM at the single-cell level should facilitate the understanding of the cellular heterogeneity and dynamics in physiological GC reactions but also may offer novel insights into the AID-dependent shaping of the genomic landscape of germinal center B-cell neoplasms.

Efficacy and Safety of Polatuzumab-Vedotin Plus Rituximab, Cyclophosphamide, Doxorubicin and Prednisone (Pola-R-CHP) for Diffuse Large B-Cell Lymphoma in the Real-World Setting: A Single Institute Retrospective Study

Background In the POLARIX study, Pola-R-CHP showed significant improvement in progression free survival (PFS) in previously untreated diffuse large B-cell lymphoma (DLBCL) compared to R-CHOP. Pola-R-CHP was approved in September 2022 in Japan ahead of the the world. However, there are still few reports about the safety and efficacy of Pola-R-CHP in real-world setting, leaving some questions about the optimal patient population for Pola-R-CHP. So, we conducted this single institute retrospective observational study in clinical practice. Methods The Pola-R-CHP group included untreated DLBCL patients who received Pola-R-CHP therapy from September 2022 to January 2023 at Kansai Medical University Hospital. The control group included untreated DLBCL patients treated with R-CHOP or R-THP-COP from January 2020 to June 2022 at our hospital. The primary endpoint was the complete remission rate (CRR) at the end of treatment, and secondary endpoints were 6-month PFS, 6-month overall survival (OS) survival, and severity and frequency of adverse events. Results In the Pola-R-CHP group, 30 patients (median age 74 [range;42-88] years) were enrolled. 8 patients over 80 years old were included. 46.7% (14/30) of the cases were Ann Arbor Stage Ⅲ-Ⅳ, and 73.3% (22/30) of the cases had IPI(International Prognostic Index) of 2 or more points. 60.0% (18/30) of patients were GCB (germinal center B-cell) type and 33.3% (10/30) were non-GCB type, and 36.7% (11/30) of patients were DEL (Double expressor lymphoma).In the control group, 100 patients (median age 75 [range;39-88] years) were enrolled. 23 patients over 80 years old were included. 54% (54/100) of the cases were Stage Ⅲ-Ⅳ, and 74%(74/100) of the cases had IPI of 2 or more points. 44%(44/100) of patients were GCB type and 47% (47/100) were non-GCB type (nine were not classified), and 40% (40/100) of patients were DEL. 65% (65/100) of patients were treated with R-CHOP and 35% (35/100) patients were treated with R-THP-COP. There were no significant differences in the patient backgrounds between the respective groups.In Pola-R-CHP cohort, the median follow-up was 282 days (range, 181-348 days). The median number of Pola-R-CHP cycles performed 6 (range; 2-6), with a 6-cycles completion rate of 86.7% (26/30). In patients over 80 years old, the doses of CHP were reduced to 50%. In the Pola-R-CHP group at end of treatment (EOT), the overall response rate was 93.3% (n=28), CRR was 86.7% (n=26), partial response was 6.7% (n=2), and progressive disease was 6.7% (n=2)(Fig.1). In the control group, CRR at EOT was 76.0% but the difference between each group was not significant (p=0.321). The PFS at 6 months was 93.3% (95% confidence interval (CI),75.9%-98.3%) in the Pola-R-CHP group and 80.0% (95%CI,70.7%-86.6%) in the control group, with significantly improvement in the Pola-R-CHP group (p=0.017). Notably, in patients with IPI of 4 to 5, CRR was 88.9 % in the Pola-R-CHP group and 51.5% in the control group (p=0.060), and the PFS at 6 months was significantly higher in the Pola-R-CHP group (88.9% [95%CI,43.4%-98.4%] vs. 60.6%[95%CI,42.0%-74.9%]; p=0.034) In the patients with high metabolic tumor volume (MTV) measured based on PET-CT (cut off value; 166 cm 3 in the top quartile of MTV), PFS at 6 months was significantly higher in the Pola-R-CHP group than the control group (100% vs 73.7% [95% CI,47.9%-88.1%]; p=0.045). OS at 6 months was 89% (95%CI,81%-93.8%) in the control group, while all patients in the Pola-R-CHP group were alive. The most common AE was hematological toxicity: neutropenia, anemia, and thrombocytopenia at grade 3-4 was observed in 43.3%, 16.7%, and 20.0%, respectively (Fig. 2). 14 cases of infection were observed. Peripheral neuropathy was found in 23.3% of the patients, and one of the patients reached grade 3-4. Many cases were complicated with constipation, but only a few cases were grade 3-4. Diarrhea was present in 20%, but all were mild and transient. No deaths due to AE were observed. One patient had to discontinue treatment due to heart failure. Unexpected adverse events were not observed. Conclusion In this study, Pola-R-CHP showed a treatment response and PFS benefit compared to historical control in patients with previously untreated DLBCL in the real-world setting.The incidence of diarrhea and peripheral neuropathy was lower, while AE profiles were similar to POLARIX study. Pola-R-CHP is well tolerated and the efficacy can be promising compared to R-CHOP.

Linperlisib in Conjunction with Gemcitabine and Oxaliplatin in Relapsed/Refractory Diffuse Large B-Cell Lymphoma: A Multicenter, Single-Arm Phase Ib/II Study

Background: Diffuse large B-cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin lymphoma (NHL), comprising approximately 30-40% of new NHL cases. Although immunochemotherapy is the current front-line treatment for DLBCL, nearly 40% of patients will face disease relapse or refractoriness to the initial therapy, with a suboptimal prognosis. Gemcitabine in combination with oxaliplatin (GEMOX) offers an established treatment regimen with a manageable side-effect profile, thus providing a viable backbone for innovative treatment combinations. Linperlisib, a selective PI3Kδ inhibitor, has demonstrated its ability to inhibit the growth of PI3Kδ-expressing human tumor cells, as evidenced by both in vitro and in vivo studies. The tolerability, pharmacokinetics, and preliminary efficacy of linperlisib were affirmed in a preceding phase I study, laying the groundwork for its further exploration in patients with relapsed or refractory (r/r) DLBCL. This study aimed to investigate the efficacy and safety of combining linperlisib with GEMOX in treating r/r DLBCL patients. Methods: In this multicenter, single-arm phase Ib/II trial (NCT04500561), histologically confirmed r/r DLBCL patients across six Chinese centers were enrolled and received oral linperlisib (80 mg/day) alongside intravenous GEMOX (gemcitabine 1000 mg/m 2 on day 1, and oxaliplatin 100 mg/m 2 on day 2) every three weeks, with GEMOX administered for a maximum of six cycles. The primary endpoint was the objective response rate (ORR), with secondary outcomes including disease control rate (DCR), time to response (TTR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Results: Between September 2020 and June 2021, 39 patients were enrolled and received the study treatment, all of whom were included in the efficacy and safety analysis. By the data cutoff in February 2023, and after a median follow-up of 24.1 months, three patients were still on treatment. The ORR reached 53.8% (95%CI: 37.2, 69.9), and the DCR was recorded at 64.1% (95%CI: 47.2, 78.8) (Figure 1). The median TTR was 1.3 months (range: 1.2-4.0). The median DOR stood at 5.7 months (95%CI: 4.3-9.1), and the median PFS was 5.4 months (95%CI: 1.8-6.7). The median OS had not yet been reached by the cutoff, with the one-year OS rate standing at 65.5% (95%CI: 48.1-78.3). Stratification by subtype revealed an ORR of 66.7% (6/9) among germinal center B-cell like (GCB) DLBCL patients compared to 40% (10/25) among non-GCB DLBCL patients. Median PFS was reported at 3.0 and 3.4 months for GCB and non-GCB DLBCL patients, respectively. The most prevalent grade 3 or higher treatment-related adverse events included decreases in neutrophil count (46.2%), platelet count (41.0%), and white blood cell count (28.2%). Conclusion: The combination of linperlisib with GEMOX exhibits a favorable safety profile and promising clinical efficacy in patients with r/r DLBCL who have received a median of two prior systemic therapies. These findings support the potential role of linperlisib combined with GEMOX as an active and safe therapeutic option for patients with r/r DLBCL, and set a foundation for further research into linperlisib's potential in combination with other treatment regimens.

YAP1-Dependent Promotion of Downstream Transcription through BRD4 Mediates Selinexor Resistance in Diffuse Large B-Cell Lymphoma

Background: Diffuse large B-cell lymphoma (DLBCL) represents the most prevalent subtype of non-Hodgkin lymphoma. Despite advancements in treatment, the prognosis for relapsed and/or refractory DLBCL (R/R DLBCL) patients remains unfavorable due to the development of drug resistance and disease progression. The pressing need to enhance the outcomes in R/R DLBCL patients necessitates ongoing investigations into novel therapeutic approaches. Among various exportins, the nuclear export protein exportin 1 (XPO1) solely governs the transportation of nuclear proteins harboring leucine-rich hydrophobic nuclear export signals (NES), including essential regulators like p53, RB1, and p27. Selinexor, an oral inhibitor specifically targeting XPO1, has emerged as a promising candidate in R/R DLBCL patients. Encouraging findings from a phase II clinical trial demonstrate a more favorable efficacy of Selinexor in the germinal center B-cell (GCB) subtype compared with that in other subtypes of DLBCL. Nonetheless, the clinical implementation of Selinexor has led to the emergence of acquired resistance, posing a significant challenge for medical practitioners. Efforts to overcome this resistance and establish strategies to maximize Selinexor's therapeutic potential remain of paramount importance in addressing the urgent clinical needs of R/R DLBCL patients. Methods: Characterization of Selinexor resistance targets was performed using a CRISPR/Cas9 screen transcription factors library pool at 0-day and 14-day time points after treatment. Cell function experiments in vitro were applied to verify the screening results and therapeutic efficacy was tested in vivo in NSG mice. Synergistic treatment approach utilizing BRD4 inhibitor JQ1, known for its curative effects in AML, MM, and ALL, in combination with Selinexor were also evaluated. Mechanistic exploration of BRD4's role in Selinexor resistance was further analyzed via RNA-seq, ChIP-seq, as well as luciferase reporter experiments. Therapeutic outcomes were evaluated in R/R DLBCL patients undergoing Selinexor treatment via PET/CT imaging. Results ： BRD4, BRD7, ZMYND8, and YTHDC1 were identified as top Selinexor resistance candidate targets through CRISPR/Cas9 Screen. The knock-down of BRD4 enhanced the killing ability of Selinexor in vitro and reduced tumor burden in subcutaneously transplanted tumor mice after treatment with Selinexor in vivo in DLBCL. The combined treatment with BRD4 inhibitor JQ1 and Selinexor had a stronger ability to promote cell apoptosis, enhance DNA damage and reduce nascent DNA compared with the single drug group. Hematological and organ toxicity tests confirmed an acceptable toxicity for the combined drug regimen. ChIP-seq differential peaks and RNA-seq differential genes analysis elucidated the crucial role of YAP1's sub-cellular localization under Selinexor treatment. YAP1 nuclear translocation enhanced transcription and formed an interaction with BRD4, resulting in augmented BRD4 binding domain, and finally lead to the resistance of Selinexor. PET/CT imaging showed that one patient with high XPO1 expression achieved complete response (CR), exhibiting a sensitivity to Selinexor treatment, but the other exhibits resistance associated with a lack of XPO1 expression and BRD4-regulated acetylation. Conclusions: The nuclear translocation of YAP1 and its interaction with BRD4 mediated the transcriptional regulation and further induced resistance to Selinexor in DLBCL. BRD4 inhibitor JQ1 combined with low-dose Selinexor can mitigate blood toxicity and enhance therapeutic efficacy, achieving an improved outcome.

Modeling the crosstalk between malignant B cells and their microenvironment in B-cell lymphomas: challenges and opportunities.

B-cell lymphomas are a group of heterogeneous neoplasms resulting from the clonal expansion of mature B cells arrested at various stages of differentiation. Specifically, two lymphoma subtypes arise from germinal centers (GCs), namely follicular lymphoma (FL) and GC B-cell diffuse large B-cell lymphoma (GCB-DLBCL). In addition to recent advances in describing the genetic landscape of FL and GCB-DLBCL, tumor microenvironment (TME) has progressively emerged as a central determinant of early lymphomagenesis, subclonal evolution, and late progression/transformation. The lymphoma-supportive niche integrates a dynamic and coordinated network of immune and stromal cells defining microarchitecture and mechanical constraints and regulating tumor cell migration, survival, proliferation, and immune escape. Several questions are still unsolved regarding the interplay between lymphoma B cells and their TME, including the mechanisms supporting these bidirectional interactions, the impact of the kinetic and spatial heterogeneity of the tumor niche on B-cell heterogeneity, and how individual genetic alterations can trigger both B-cell intrinsic and B-cell extrinsic signals driving the reprogramming of non-malignant cells. Finally, it is not clear whether these interactions might promote resistance to treatment or, conversely, offer valuable therapeutic opportunities. A major challenge in addressing these questions is the lack of relevant models integrating tumor cells with specific genetic hits, non-malignant cells with adequate functional properties and organization, extracellular matrix, and biomechanical forces. We propose here an overview of the 3D in vitro models, xenograft approaches, and genetically-engineered mouse models recently developed to study GC B-cell lymphomas with a specific focus on the pros and cons of each strategy in understanding B-cell lymphomagenesis and evaluating new therapeutic strategies.

Tumor Mutational Burden As a Prognostic Factor in Diffuse Large B-Cell Lymphoma

Background: Tumor mutational burden (TMB), defined as the number of nonsynonymous mutations per megabase (Mb) in the coding regions of a tumor genome, has been associated with improved response to immune checkpoint blockade treatments and more broadly with overall survival (OS) in many cancers. Limited prior studies have found that TMB is higher in diffuse large B-cell lymphoma (DLBCL) compared to many other cancers. However, only two studies have evaluated TMB and prognosis in newly diagnosed DLBCL patients. The prognostic role of TMB in diffuse large B-cell lymphoma (DLBCL) remains unclear as prior studies have been limited to panel sequencing of cancer genes and/or lacked matched germline-tumor samples for filtering. Methods: We characterized TMB from whole exome sequencing (WES) in newly diagnosed DLBCL patients with matched diagnostic tumor-normal samples prior to treatment with standard immunochemotherapy (IC). Tumor DNA was extracted from formalin-fixed, paraffin embedded tissue sections and germline DNA from peripheral blood. We evaluated the association between TMB and event-free (EFS) and OS using Cox regression adjusted for the international prognostic index (IPI) and other factors including cell of origin (COO), MYC double hit (DH) with BLC2 or BCL6 by FISH, and TP53 mutation status. EFS and OS were defined as time from diagnosis to earliest of treatment failure (EFS only), death, or last follow-up; EFS at 24 months (EFS24) was also modeled as a marker of early aggressive DLBCL. An optimal high/low TMB cutpoint was determined based on EFS using maximally selected rank statistics; sensitivity analyses used an 20/80 percentile to define the threshold. We also evaluated the association of driver tumor mutational load (driverTML) with EFS and OS, defined as the total number of mutations in 292 DLBCL driver genes. Results: In the analysis cohort of 394 DLBCL patients, the median age at diagnosis was 65 years (interquartile range: 56-73), 43% were female, 62.2% had IPI 0-2, and 57.4% germinal center B-cell COO. MYC was DH in 7.5% and TP53 was mutated in 16.2% of patients. Through a median follow-up of 7.5 years, there were 141 events and 122 deaths. EFS24 was achieved in 75.1% of the cohort; 5-year EFS was 69% (95% CI 64-74%) and 5-year OS was 75% (95% CI 71-80%). Median TMB in our cohort of DLBCL patients was 3.6 mutations/Mb, which was the fifth highest compared with 32 cancers in The Cancer Genome Atlas. As a continuous variable, increasing TMB (mutation/Mb) was associated with superior EFS (HR=0.89, 95% CI 0.81-0.98) and OS (HR=0.89, 95% CI 0.81-0.99) after adjustment for IPI. Spline analyses confirmed linear associations of TMB with EFS and OS. The optimal modeled cutpoint based on EFS was 4.5 mutations/Mb, with 31% classified as high TMB (>4.5). No significant ( P >.05) differences were observed between high and low TMB patients with clinical (i.e., age, sex, IPI category) or molecular (cell of origin, MYC DH, TP53 mutated) factors. In Cox regression, high-TMB was significantly protective for EFS and for OS compared to low-TMB after adjustment for each clinical factor ( Figure 1), with simultaneous adjustment for all these factors associated with superior EFS (HR=0.52, 95% CI 0.33-0.84) and OS (HR=0.59, 95% CI 0.36-1.00). TMB (high/low) combined with IPI (0-2, 3-5) was significantly ( P =.001) associated with EFS in Kaplan-Meier analysis ( Figure 2). Among patients with IPI 3-5, high TMB was significantly associated with superior EFS (HR=0.47, 95% CI 0.27-0.81) and OS (HR=0.57, 95% CI 0.33-0.99). Among patients with IPI 0-2, we also observed longer EFS (HR=0.62, 95% CI 0.34-1.11) and OS (HR=0.62, 95% CI 0.32-1.20) in high TMB patients, although the associations were not statistically significant ( P >.05). In sensitivity analyses, a 20/80 percentile TMB threshold found similar results for EFS (HR=0.64, 95% CI 0.41-1.03) and OS (HR=0.69, 95% CI 0.42-1.12) as in our main analyses. TMB and driverTML were significantly correlated ( R=0.65, P <10 -16) and we found similar effect sizes for EFS (HR=0.45, 95% CI 0.22-0.91) and OS (HR=0.55, 95% 0.27-1.13) as in our main analyses. Conclusions: In a large and well-defined cohort of IC treated DLBCL patients, we found DLBCL patients with higher TMB had superior EFS and OS after adjustment for other clinical and molecular prognostic factors, and the prognostic association for TMB was robust to threshold and gene set.

MYC to BCL6 State-Transitions Determine Cell Size and Metabolic Fluctuations and Define a Novel Biorhythm in B-Cell Malignancies

Background: Cell size fluctuations are well-documented during critical transitions during normal B-cell development. For instance, during early stages of development, “large cycling pre-B cells” (also classified as Fraction C') eventually exit cell cycle and shrink to become “small resting pre-B cells” (Fraction D) to enable immunoglobulin light chain gene recombination. We and others had shown that this transition is marked by loss of Myc expression and gain of Bcl6. Later in development, germinal center B-cells cycle between large MYC+ “centroblast” and smaller BCL6+ “centrocyte” states between dark and light zones, respectively. MYC is involved in biomass accumulation and provides fuel for cell division, while BCL6 confers a quiescent phenotype to cells and protects B cells from DNA damage-induced apoptosis. BCL6 can suppress MYC transcription, indicating MYC and BCL6 is mutually exclusive, and their expression dictates distinct cellular states. Significance: In B-cell malignancies, transitions between mutually exclusive MYC+ and BCL6+ states seem to be important as well: B-ALL cells that are driven by oncogenic tyrosine kinases (e.g. BCR-ABL1) express predominantly MYC but can be forced into a BCL6+ state upon tyrosine kinase inhibition (e.g. imatinib). In germinal center-derived B-cell lymphoma, both MYC and BCL6 are frequently targeted by chromosomal translocation, suggesting that disruption of physiological state transitions may be part of the malignant transformation program. Results: To elucidate regulation of MYC-BCL6 state transitions and their importance in progression and development of drug resistance in B-ALL and germinal center-derived B-cell lymphoma, we developed a MYC-eGFP and BCL6-mCherry dual-reporter mouse model. In addition, we engineered human B-ALL PDX cells with MYC-mNeonGreen and BCL6mScarlet knockin fusion genes by CRISPR/Cas9-mediated HDRT. As expected, MYC+ and BCL6+ states were largely mutually exclusive during early stages of B-cell development, as well as in BCR-ABL1- and NRAS G12D-driven B-ALL. Interestingly, a large fraction of B-ALL cells expressed neither MYC nor BCL6, as validated by cell sorting and Western blot. Based on single-cell sorting and subsequent time-lapse monitoring over 12 hours, we found MYC+ cells transitioning through a double-negative state to become BCL6+ and then revert again to a MYC+ state ( Figure A), suggesting dynamic transitions between MYC and BCL6 cycles. We characterized these populations by analyzing cell size, cell proliferation, cell cycle, clone formation, and gene expression profiles. The results indicated that MYC+ cells exhibited larger cell size, active proliferation, and increased glycolytic activity. Conversely, BCL6+ cells displayed smaller cell size, activation of autophagy with suppression of glycolysis, and cell cycle arrest in the G0 phase of the cell cycle. Conclusions and future directions: Given the dynamic inter-transition between MYC and BCL6 states, and the significant roles of both MYC and BCL6 in B-ALL and germinal center-derived B-cell lymphomas, we propose MYC- and BCL6-dependent fluctuations of cellular activity (wake) and quiescence (sleep; Figure B). Thereby, MYC and BCL6 are expressed in an oscillatory manner. During the MYC state, cells exhibit high glycolysis activity, actively accumulate biomass, and undergo proliferation, representing the wake phase. In contrast, during the BCL6 state, cells suppress glycolysis metabolism, pause the cell cycle, and display a quiescent phenotype, representing the sleep phase. Based on this discovery, we will examine the ‘B-cell exhaustion’ paradigm that Bcl6 and Myc mark iterative cycles of quiescence and activation. In analogy to sleep-wake phases, we hypothesize that the Bcl6-autophagy phase is essential for recovery and regeneration. We will investigate how B-cells transition from one phase to the next and consequences of elongation of one phase at the expense of the other. Malignant B-cells may be particularly sensitive to shortening of quiescence periods (“sleep deprivation”). These and other observations will lead to new conceptual frameworks for the understanding of how MYC-BCL6 state transitions, cell-size fluctuations and the length of recovery-periods regulate energy-supply and survival of B-cell malignancies and create opportunities for therapeutic intervention.

CDCA2 Is a Predictive Marker and Mediator of Bortezomib Response in Diffuse Large B-Cell Lymphoma

Objective: In recent years randomized phase III clinical trials have attempted to improve first-line R-CHOP treatment of diffuse large B-cell lymphoma (DLBCL) through addition or substitution of drugs. Bortezomib treatment has shown significant clinical efficacy in other B-cell malignancies, and the REMoDL-B trial testing R-CHOP vs. RB-CHOP in DLBCL patients stratified by cell of origin revealed at 60 months follow-up that ABC and molecular high-grade patients significantly benefitted in clinical outcome. This study aims at identifying predictive markers of bortezomib response combined with functional investigation of their biological impact in DLBCL irrespective of molecular subtypes. Methods: Data from the REMoDL-B trial including 450 patients in each treatment arm was retrospectively analyzed. We fitted a multivariate Cox regression model testing all expressed genes with overall survival as outcome and an interaction term between gene expression and treatment arms (R-CHOP and RB-CHOP) as covariates, including their main effects. For the top candidate, CDCA2, survival was visualized using Kaplan-Meier analysis of patients stratified by median expression. Functional examination of CDCA2 was performed by applying CRISPR/Cas9 genome editing. Knockout of CDCA2 was conducted in two DLBCL cell lines, representing ABC and GCB cell of origin, and effect on proliferation, cell cycle distribution, and drug response was examined in MTS-assays and by flow cytometry. A DLBCL xenograft mouse model was established with CDCA2 knockout and control DLBCL cells, respectively, and time to tumor formation and death was monitored and tumors molecularly characterized. Results: DLBCL patients with high expression of CDCA2 have superior outcome when treated with RB-CHOP in comparison to R-CHOP (p=0.0097), whereas no difference in outcome was observed for patients with low CDCA2 expression (p=0.086) (Figure 1), supporting high expression of CDCA2 as a predictive marker of bortezomib response irrespective of cell of origin subclassification. Knockout of CDCA2 decreased DLBCL cell proliferation rate and affected the cell cycle transition between the G1 and S-phase, resulting in an increased percentage of cells in G1 compared to the scramble control. Bortezomib dose-response analysis revealed reduced sensitivity in CDCA2 knockout cells compared to the control (Figure 2A). In addition, CDCA2 knockout cells were also less sensitive to carfilzomib, a 2 nd generation proteasome inhibitor. In combinatory drug experiments comparing CHOP and B-CHOP, CDCA2 knockout cells demonstrated higher relative resistance compared to controls cells when exposed to B-CHOP than CHOP (Figure 2B+C), illustrating CDCA2 to be cardinal and specifically mediating the bortezomib response. In DLBCL xenograft models, CDCA2 knockout tumors displayed reduced growth rate in comparison to control tumors. Due to the substantial tumor burden, control DLBCL xenografted mice had shorter survival time than CDCA2 knockout DLBCL mice (Figure 2D). Conclusion: DLBCL patients with high expression levels of CDCA2 benefitted the most from addition of bortezomib to R-CHOP. In functional studies, the loss of CDCA2 increased cellular resistance to bortezomib in both single- and combinational drug studies.

α-Glucosidase inhibitors boost gut immunity by inducing IgA responses in Peyer's patches.

Peyer's patches (PPs) are specialized gut-associated lymphoid tissues that initiate follicular helper T (Tfh)-mediated immunoglobulin A (IgA) response to luminal antigens derived from commensal symbionts, pathobionts, and dietary sources. IgA-producing B cells migrate from PPs to the small intestinal lamina propria and secrete IgA across the epithelium, modulating the ecological balance of the commensal microbiota and neutralizing pathogenic microorganisms. α-glucosidase inhibitors (α-GIs) are antidiabetic drugs that inhibit carbohydrate digestion in the small intestinal epithelium, leading to alterations in the commensal microbiota composition and metabolic activity. The commensal microbiota and IgA responses exhibit bidirectional interactions that modulate intestinal homeostasis and immunity. However, the effect of α-GIs on the intestinal IgA response remains unclear. We investigated whether α-GIs affect IgA responses by administering voglibose and acarbose to mice via drinking water. We analyzed Tfh cells, germinal center (GC) B cells, and IgA-producing B cells in PPs by flow cytometry. We also assessed pathogen-specific IgA responses. We discovered that voglibose and acarbose induced Tfh cells, GCB cells, and IgA-producing B cells in the PPs of the proximal small intestine in mice. This effect was attributed to the modification of the microbiota rather than a shortage of monosaccharides. Furthermore, voglibose enhanced secretory IgA (S-IgA) production against attenuated Salmonella Typhimurium. Our findings reveal a novel mechanism by which α-GIs augment antigen-specific IgA responses by stimulating Tfh-GCB responses in PPs, and suggest a potential therapeutic application as an adjuvant for augmenting mucosal vaccines.