GCB Diffuse Large B-cell Lymphoma Research Articles

Polatuzumab vedotin in diffuse large B-cell lymphoma therapy: Literature review and experience of the Oncohematology Division of the Lapino Clinical Hospital

Study of molecular and genetic features of diffuse large B-cell lymphoma (DLBCL) suggests differential approach to treatment of this aggressive B-cell disorder characterized by short remission after each subsequent therapy line. Therefore, more effective regimens should be used at earlier stages. Identification of biological markers and their integration into prognostic scales can help to personalize therapy, especially in patients with high risk of quick progression and increase survival.Our own experience of using immunoconjugate polatuzumab vedotin in therapy of patients with DLBCL. The use of Pola-BR combination in a patient with refractory non-GCB DLBCL allowed to achieve full remission alongside satisfactory tolerability and absence of significant adverse events. An evaluation of the Pola-R-CHP regimen in treatment of a patient with newly diagnosed generalized GCB DLBCL is presented.

Abstract 3913: Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy

Abstract Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas. However, the mechanisms by which GCs kill malignant lymphoma cells still largely remains elusive due to the pleiotropic consequences of GC binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. This prompted us to search for critical direct targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To determine effector genes that increase glucocorticoid toxicity, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone. Screens in cell line models of Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor (BCR) signaling pathway (≥2 SD). To identify direct transcriptional GR targets and binding sites genome-wide, we performed the cleavage under targets and release using nuclease (CUT&RUN) assay and integrated this dataset with RNA-seq upon GC treatment. The pairwise comparison analysis (p<0.05) for GR-bound genes across BL and DLBCL cell lines revealed a significant enrichment of negative regulators of BCR/PI3K signaling and BCR-related tumor suppressors (LAPTM5, KLHL14, ARID5B, DDIT4, DAPP1, INPP5D, and CSK). Interestingly, expression of CSK, an inhibitor of all Src-family kinases (SFKs), was repressed while that of other BCR negative regulators were directly activated by binding of GR. Combinatorial deletion of activated genes counteracted the toxicity of GC up to 83.9%. In contrast, depletion of CSK paradoxically increased the cytotoxic effects of GC in BCR-dependent aggressive lymphoma with decreased proximal BCR signaling. To gain further insights on CSK function in aggressive lymphomas, we performed phospho-proteome and ubiquitinome profiling by treating lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi). The CSKi treatment initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-κB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs. Furthermore, small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers. Citation Format: Jaewoo Choi, Michele Ceribelli, James D. Phelan, Björn Häupl, Da Wei Huang, George W. Wright, Tony Hsiao, Vivian Morris, Francesco Ciccarese, Boya Wang, Sean Corcoran, Sebastian Scheich, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Joyce Zhou, Grace Zhang, Jagan Muppidi, Giorgio Ga Inghirami, Thomas Oellerich, Craig J. Thomas, Wyndham H. Wilson, Louis M. Staudt. Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3913.

Abstract 463: The small nucleolar RNA SNORD46 and SNORD72 have tumor suppressor activity in diffuse large B cell lymphoma

Abstract Background. Following the identification of an 11q24.3 gain upregulating the ETS factors ETS1 and FLI1 in diffuse large B cell lymphoma (DLBCL) (Bonetti et al. Blood 2013), we observed the anti-lymphoma activity of blocking the protein-protein interaction of ETS factors with RNA helicases, using YK-4-279 and its clinical derivative TK-216 (Spriano, Chung et al. CCR 2019). DLBCL cells exposed to the two small molecule inhibitors presented a significant upregulation of small nucleolar RNAs (snoRNAs). Besides their primary functions in rRNA processing, snoRNAs are often deregulated in cancers, with microRNA-like actions and direct interaction with proteins. Here, we characterized SNORD46 and SNORD72, the two snoRNAs most upregulated cells after YK-4-279 and TK-216 and mapped to the 1p34.1 locus recurrently lost in DLBCL. Methods. Real-time PCR, immunoblotting, and shRNA silencing were conducted based on established protocols. RNA-Protein Interaction Prediction (RPISeq) was used to calculate the interaction probabilities of snoRNAs with proteins of interest. The prediction was made using both the Random Forest (RF) and the Support Vector Machine (SVM) classifiers, using a stringent threshold of >0.6. Results. The time-dependent changes of SNORD46 and 74 were validated with real-time PCR in the U2932 activated B cell-like (ABC) DLBCL cell line exposed to YK- 4-279 for 4 or 8 h. Upregulation of SNORD46 and 74 were significantly upregulated compared to their host genes upon YK-4-279 treatment, suggesting these snoRNAs have different functions from the genes in which they are encoded. Across 48 lymphoma cell lines, SNORD74 was more expressed in ABC DLBCL than in the remaining cell lines (P 0.03) or GCB DLBCL (P 0.05), while SNORD46 did not differ. Stable lentiviral-based overexpression of SNORD46 and 74, U2932 ABC DLBCL demonstrated that both SNORD46 and SNORD74 overexpression impaired growth while SNORD92, the control did not SNORD74 overexpression inhibited pAKT-Ser473, suggesting it could exert its anti-tumor effect by negatively impacting BCR signaling. Since constitutive BCR is the main driver for the pathogenesis in ABC DLBCL, we studied the impact of BCR signaling on SNORD46 and 74 expressions. IgM stimulation downregulated the snoRNAs in U2932 and TMD8 ABC DLBCL cell lines at 1 and 2.5 h poststimulation, with a more substantial effect for SNORD74. Furthermore, the snoRNAs were upregulated in four ABCs and four GCB DLBCLs cell lines exposed to the PI3K/mTOR inhibitor bimiralisib. Bioinformatic analysis indicated the possible binding of SNORD46 and especially of SNORD74 to BCR signaling molecules (SYK, LYN, BTK, PI3Kδ, IRAK4, IRAK1, CARD11), RNA helicases (RHA, DDX21, DDX5) and essential DLBCL proteins (BCL6, IRF4). Conclusions. SNORD46 and SNORD74 exert tumor suppressor activity in DLBCL cells, possibly as negative regulators of the BCR signaling. Citation Format: Elaine YL Chung, Filippo Spriano, Sara Napoli, Giulio Sartori, Luciano Cascione, Francesco Bertoni. The small nucleolar RNA SNORD46 and SNORD72 have tumor suppressor activity in diffuse large B cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 463.

A Multi-Omic Study Unveils a Clonal Population of TET2-Mutated Infiltrating T-Cells in Germinal Center B Cell Lymphomas (DLBCL and FL)

Introduction Follicular lymphomas (FL) and diffuse large B cell lymphomas (DLBCL) share a common germinal center (GC) B cell precursor. Nevertheless, these lymphomas appear to originate from different developmental stages, through distinct pathogenetic mechanisms and their clinical behavior varies significantly, even among FL or DLBCL patients. To understand the molecular and transcriptional heterogeneity underlying this clinical diversity we conducted a multi-omic study in GC B cell lymphomas. Methods Our study comprised 3 DLBCL patients (1 non-GCB (germinal center B), 1 non-GCB CD5+, and 1 GCB) and 2 FL (1 with no transformation (ntFL) and 1 patient with transformation (tFL)) at diagnosis. We performed single-cell multiome-sequencing (scDNA-seq + scProtein) ( Tapestri platform -Mission Bio-) and single cell RNA-sequencing (scRNA-seq) ( Chromium system -10xGenomics-) from cell suspensions and spatial transcriptomics ( Visium technology -10xGenomics-) from paraffin blocks. Results Genotyping data were available for 19,700 cells. A total of 311 variants across the 5 samples passed all quality controls. We selected probably pathogenic and pathogenic variants for downstream analysis. Germline variants were discarded. In 3 of 5 patients, we detected 1 somatic and nonsynonymous variant defining the first clone (variants in KMT2D and NOTCH2 mutated in 6-38% of the cells) and another somatic and nonsynonymous variant, defining a subclone (variants in KMT2D, ATM, EZH2 mutated in 1-15% of the cells). In 1 of 5 patients (ntFL), we detected up to 4 mutations, acquired linearly ( B2M, GNA13, EZH2, RHOA mutated clone represents 8% of the cells). Remarkably, all patients showed the same pathogenic variant in TET2 (1-2% of the cells), as an independent clone. The highest molecular similarity was observed between the GCB DLBCL patient and the tFL patient. Both had the same variant in KMT2D as the first hit (later GCB-DLBCL acquired ATM, while tFL acquired EZH2 as a second hit). ATM and EZH2 mutations have been described in “bulk” studies conducted on DLBCL and FL patients, respectively. Regarding protein expression, there was a good correlation between GCB and non-GCB phenotypes studied by immunohistochemistry and scProtein (the expression of CD10 by proteomics was lower in non-GCB patients). TET2 clones described by scDNA-seq were enriched in CD5+ and CD19- cells, suggesting they belong to the T-lymphoid lineage. The other clones and subclones were enriched in CD19+ and CD5- cells ( Figure 1). Data from 54,024 single cells were obtained for scRNA-seq analysis, with an average of 10,805 cells (7,335-18,205) for each sample. Cell type annotation was performed based on the expression of canonical markers. We identified 13 different clusters, the main ones being: GC B cells, post-GC B cells, naïve B cells, myeloid cells, CD8+ cytotoxic T cells, CD4+ naïve T cells, CD4+ reg T cells, CD4+ helper T cells. All samples contained subpopulations of both GC and post-GC B cell clusters. However, GCB DLBCL and FL patients were significantly enriched in GC B cells (63.2 vs 22.2%; p<0.01), whereas non-GCB patients were significantly enriched in post-GC B cells (35.5 vs 2.9%; p<0.01). Part of the normal B-cell transcriptional function was preserved in all samples ( Figure 2). The analysis of the infiltrating T cells revealed that the CD4+/CD8+ T cells ratio progressively increased from ntFL (1.14) to tFL (2.25) and DLBCL (2.58). Furthermore, we estimated the exhaustion status of T cells by the expression of these five markers ( TIGIT, LAG3, CTLA4, HAVCR2, PDCD1). Exhaustion T cell markers were highly expressed in CD8+ cytotoxic T cells and cycling T cells and lower expressed in CD4+ reg T cells. Both DLBCL and tFL have a stronger pattern of exhaustion than ntFL. Finally, by the spatial transcriptomics analysis, we have identified distinct cell type proportions according to their matched single-cell RNA data. Conclusions Our results confirm the intra and intertumor heterogeneity in GC B cell lymphomas. Notably, the greatest molecular and transcriptional similarities are observed between the GCB DLBCL and tFL patients at diagnosis. Moreover, our findings support the critical role of the tumor microenvironment for the persistence and development of the tumor clone. The finding of a TET2 mutated clone in the infiltrating T cells of all the patients is particularly noteworthy, as it may suggest the possible presence of clonal lymphopoiesis.

Molecular Mechanism of Action of Glucocorticoids in Lymphoma Therapy

Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas, including diffuse large B cell lymphoma (DLBCL) and Burkitt lymphoma (BL). GCs act by binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. The transcriptional response to GCs can vary significantly due to the variation in expression levels of GR cofactors and chromatin landscapes in different cell types. The mechanisms by which GCs kill malignant lymphoma cells are largely unknown, prompting us to search for new targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To identify genes that synergize or antagonize GC lethality in lymphomas, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone, the active metabolite of prednisone. Screens in cell line models of BL and DLBCL (both ABC and GCB subtypes) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor signaling pathway, which is required to sustain the viability of these malignant lymphoma cells. With combination of the cleavage under targets and release using nuclease (CUT&RUN) assay and RNA-seq, we identified GR binding to the LAPTM5 locus at its glucocorticoid response element and AP1 motifs upon GC treatment. GR binding also induced expression of LAPTM5, which negatively regulated BCR signaling by promoting the lysosomal degradation of the BCR. Conversely, GC induced binding of GR to the CSK locus, thereby repressing expression of the non-receptor tyrosine kinase CSK, which antagonizes BCR signaling by phosphorylating an inhibitory tyrosine residue present in all Src-family kinases (SFKs). However, in BCR-dependent aggressive lymphomas, inactivation of CSK paradoxically decreased proximal BCR signaling and induced cell death. By performing quantitative phosphoproteome and ubiquitinome with mass spectrometry, we demonstrated that treatment of lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi) initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-kB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs, which have been used empirically for decades to treat lymphomas. GCs restrain the most proximal steps in BCR signaling at the plasma membrane by, on one hand, decreasing BCR abundance, and on the other hand, by decreasing CSK expression, thereby reducing expression of the essential SFKs. Small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers.

An In Vivo PiggyBac Insertional Mutagenesis Screen Reveals Oncogenic Lesions Cooperating with Myd88 L265P

Diffuse large B cell lymphoma (DLBCL) is the most common Non-Hodgkin lymphoma and originates from transformed germinal center-experienced B cells. Traditionally, DLBCL has been divided into two subtypes, depending on whether the transcriptional profile of the tumor relates to an activated B cell or a germinal center B cell (ABC and GCB DLBCL, Alizadeh et al., 2000). More recent efforts classified DLBCL cases based on their genetic aberrations and identified several clusters with distinct mutational profiles (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020). The MCD/C5 cluster is characterized by recurrent mutations in MYD88, PRDM1 and frequent amplifications of BCL2, amongst others. We recently showed that mice harboring a B cell-specific Myd88 L252P mutation (orthologous position of the human p.L265P mutation) develop B cell proliferation and occasional transformation into DLBCL (Knittel et al., 2016). Lymphomagenesis is further increased when Myd88 L252P is combined with BCL2 overexpression and a genetically engineered block in plasmacytic differentiation by loss of Prdm1 or overexpression of Spib (Flümann et al., 2021). To ask which further genes and pathways cooperate with Myd88 L252P in lymphomagenesis, we performed an in vivo piggyBac insertional mutagenesis screen. In this system, a conditionally expressed transposase mobilizes transposable elements. These elements can then reintegrate into the genome and either silence or drive the expression of genes, depending on the exact integration site and orientation of the transposon cassette (Rad et al., 2015). We crossed this piggyBac system onto a Myd88 L252P background to identify genes that cooperate with Myd88 mutations in lymphomagenesis. Mice harboring both the Myd88 mutant allele and the piggyBac system lived significantly shorter than controls harboring only the Myd88 mutation or just the piggyBac system. Myd88/piggyBac animals developed B220 +/CD138 - lymphomas and DNA isolated from these lesions allowed the detection of common transposon insertion sites. Among the genes significantly enriched for integrations, we observed known genetic drivers of human MCD/C5 DLBCL, such as PIM1 and ETV6. We also identified TBL1XR1 and SPIB as common insertion sites, as well as BCL2, BIM1 and BCL-XL, further validating our approach of engineering a plasma cell differentiation block as well as anti-apoptotic BCL2 overexpression on a Myd88-mutant background to model MCD DLBCL. Of note, the hits identified in this screen were significantly distinct from hits identified to drive Myc-driven B cell lymphomagenesis (Weber et al., 2019). Additionally, we identified several candidate genes that are not reported to be frequently genetically altered in DLBCL, however associated with processes relevant to B cell biology, particularly to B cell receptor signaling. As ‘B cell receptor signaling’ was a prominent term in our screen hits and CD79B ITAM mutations are an additional hallmark of MCD/C5 DLBCL (Chapuy et al., 2018, Schmitz et al., 2018, Wright et al., 2020), we introduced a conditional Cd79b p.Y195H allele to our Cd19 Cre/wt;Myd88 cond.p.L252P/wt; Rosa26 LSL.BCL2-IRES-GFP/wt; Prdm1 fl/fl MCD/C5 DLBCL model. While we did not observe significant differences in the immunohistochemial and transcriptional phenotype of Cd79b WT and mutant lymphomas, Cd79b p.Y195H tumors showed increased B cell receptor (BCR) signaling activity, indicated by increased levels of phosphorylated SYK and PLCg2. Cd79b mutant lymphomas also showed an increased formation of cytoplasmic signaling complexes comprised of MYD88 and several components of the BCR pathway, including MALT1 and BTK. The formation of these complexes depended on active BTK, as treatment with the BTK inhibitor ibrutinib reduced complex formation to levels found in Cd79b WT lymphomas. Consequently, we investigated the effects of ibrutinib treatment in Cd79b-mutant and wildtype MCD DLBCL mouse models and found Cd79b-mutant lymphomas to be significantly more sensitive to ibrutinib treatment than their Cd79b WT counterparts.

Characterization of Hemophagocytic Lymphohistio(HLH）in Relapsed Diffuse Large B-Cell Lymphoma Patients after CAR-T Therapy

Background: Patients with chemotherapy-refractory diffuse large B-cell lymphoma (DLBCL) have poor prognoses. The objective response rate of patients with standard salvage regimens is 26%, while CART therapy have complete remission (CR) rates of 40%- 54% against DLBCL. Half of DLBCL patients relapsed or progressed after chimeric CAR-T cell therapy, but only a few of them were accompanied by HLH. Its clinical characteristics are unclear. This study aimed to investigate the clinical characteristics of these patients. Methods: We retrospectively analyzed the clinical data of 8 patients with relapsed DLBCL lymphoma accompany by HLH after CAR-T therapy between May 2019 and July 2023(NCT02537977 and NCT04036019). We investigate the clinical characteristics of these patients, compare the survival between patients with and without CR after CAR-T therapy, compare the survival between patients with and without CRS after CAR-T therapy. Results: 8 R/R DLBCL patients were enrolled. Median age was 59.5 years (range, 37-69), and 6(75%) patients had non-germinal center B-cell-like (non-GCB) DLBCL, one had GCB DLBCL, one had transformed DLBCL. 2 patients (25.0%) had HLH before CAR-T treatment previously. Among them, 3 patients (37.5%) achieved CR after CART therapy, while 3 patients (37.5%) achieved partial remission. ALL of the patients (100%) relapsed or progressed, and all of them died because of disease progression. With a median follow-up of 7 months, the median overall survival (OS) was 206.0 days (95%CI 106.2-305.8) and progress-free survival (PFS) was 125.0 days (95%CI 40.5-210.0). Particularly，the median time from HLH to death was only 45.0 days (95%CI: 22.8-67.2). 6 patients (75.0%) occurred grade 1-2 CRS, no patient occurred ≥grade 3 CRS, and 2 patients (25.0%) did not occurred any CRS. The median OS was 328.0 days and 142.0 days (95%CI 60.4-223.6) for patients without CRS and with CRS1-2, respectively. There is significantly different in OS for patients with and without CRS ( P=0.03). The median OS was 328.0 days (95%CI 217.6-438.4) and 142.0 days (95%CI 133.4-150.6) for patients with CR and without CR, respectively. The median PFS was 257.0 days (95%CI 103.4-410.6) and 77.0 days (95%CI 8.3-145.7) for patients with CR and without CR. There is significantly different in OS and PFS for patients with CR and other patients ( P=0.01 and P=0.01). Conclusion: Hemophagocytic lymphohistiocytosis (HLH) is a rare and life threatening disorder. The prognosis of patients with relapsed DLBCL lymphoma accompany by HLH after CAR-T therapy is very poor. Patients with CRS during CART therapy may more likely have HLH when DLBCL relapse. The patients without CRS have better survival time than those with CRS during CART therapy. We hypothesis that because both HLH and CRS is inflammatory syndrome resulting from T-cell activation.

BTK Inhibitors and Chidamide-Combined Therapy in Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Background: Relapsed or refractory (R/R) diffuse large B-cell lymphoma (DLBCL) shows poor clinical outcomes, particularly in patients ineligible for stem cell transplantation or who failed induction therapy or salvage therapy. Monotherapy Bruton tyrosine kinase inhibitors (BTKi) have established modest antitumor effect in R/R DLBCL by targeting B-cell receptor signaling, with median progression-free survival (PFS) and overall survival (OS) of 1.64-2.8 months and 6.4-8.4 months, respectively. Chidamide is a selective oral histone deacetylase inhibitors (HDACi) that shows promising treatment for DLBCL and may overcome BTKi resistance. So, we evaluated the efficacy and safety of BTKis and chidamide-combined therapy in R/R DLBCL. Methods: This retrospective study consists of 11 R/R DLBCL who received combination therapy of chidamide and BTK inhibitors between September 2021 and July 2023. Chidamide was orally administered at a dose of 20 mg twice a week, while the BTK inhibitor was administered daily. Patient clinical characteristics were collected upon patient enrollment. Efficacy outcomes included overall response rate (ORR), PFS and OS, whereas safety outcomes included incidence of adverse events. Results: 11 R/R DLBCL patients were enrolled. Median age was 61 years (range, 56-73), and 81.82% of the patients had non-germinal center B-cell-like (non-GCB) DLBCL, one had GCB DLBCL, one had transformed DLBCL. The median number of previous treatment lines was 3 (range, 3-8). 10 patients (90.91%) received treatment with BTK inhibitors before. Four patients（36.36%）failed to chimeric antigen receptor T-cell/NK-cell(CAR-T/CAR-NK) therapy. The best ORR was 81.82%, with 9 patients acquired objective disease remission after 1-2 cycles. Among them, 3 patients (27.27%) achieved complete response, and6 patients (54.55%) achieved partial remission. With a median follow-up of 5 months, the median PFS was 6.0 months (95%CI 3.14-8.86) and median OS was not yet reached. In terms of adverse events, the most common hematologic toxicities observed were grade ≥3 neutropenia (36.36%, 4/11) and grade ≥3 thrombocytopenia (36.36%, 4/11). Two patients (18.18%) had pneumonia, and one patient (9.09%) had herpes zosters. Due to the positive real-world outcomes observed in the retrospective study, prospective clinical studies of chidamide in combination with BTK inhibitors for the treatment of R/R DLBCL are underway (ChiCTR2300073233). Conclusion: This real-world study revealed promising results. Despite the majority of patients had been treated with BTK inhibitors and experienced relapse and resistance, the addition of chidamide brought patients back into remission, suggesting that chidamide can enhance the efficacy of BTK inhibitors and overcome acquired resistance. Notably, chidamide combined with BTK inhibitors showed high efficacy and manageable tolerability, making it a promising treatment option for patients with R/R DLBCL. Keywords: BTK inhibitors, Chidamide, R/R DLBCL, efficacy and safety

Enhanced Genomic Stability in Monomorphic Post-Transplant Lymphoproliferative Disorders Is Driven By Distinct Mechanisms Stratified By EBV Status

Introduction: We previously showed that HIV associated EBV negative (-) diffuse large B-cell lymphomas (DLBCL) of germinal center origins (GCB) have enhanced genomic stability and increased expression of DNA repair genes compared to their HIV negative counterparts (Maguire et al 2019). Given that immune deficiency can be induced virally or therapeutically, including the administration of immunosuppressive drugs post solid organ or hematopoietic stem cell transplant, we questioned whether these observations are specific to HIV or if they represent a more global immune impaired signature. Here we assessed the genomic stability of monomorphic DLBCL post-transplant lymphoproliferative disorders (PTLD) and the potential role of DNA repair. Methods: A total of 49 monomorphic DLBCL PTLDs (26 EBV(-), 23 EBV positive (+)) and 170 immune competent (IC) DLBCL (EBV status unknown) were studied. All were pathologically reviewed and samples with <60% tumor content were macro-dissected before RNA/DNA extraction. Cell of origin (COO) profiles were assessed using the DLBCL90 molecular assay which can distinguish primary mediastinal B-cell lymphoma (PMBL) from GCB, Activated B-cell (ABC) and unclassified (UNC) DLBCL (Ennishi et al 2019). NanoString gene expression profiling (GEP) was performed using the PanCancer Pathways panel and analyzed using NanoString nSolver Advanced Analysis. Copy number (CN) analysis was performed on a subset of samples (45 PTLD, 41 DLBCL) using the OncoScan CN assay and analyzed using Chromosome Analysis Suite (ChAS) v4.1. All p-values listed were Benjamini-Hochberg adjusted and Mann-Whitney tested for the GEP and CN data respectively. Results: In contrast to IC-DLBCL which are GCB enriched (50%), COO profiling revealed PTLD to be ABC enriched (43%). CN analyses revealed that the median CN/sample for PTLD (0.57, p<0.001), EBV(+)PTLD (0.25, p<0.001) and EBV(-)PTLD (0.80, p = 0.050) tumors were significantly less aberrant than IC-DLBCL (1.01). The data also showed that EBV(+)PTLD are less aberrant than EBV(-)PTLD tumors (0.25 vs 0.80, p<0.001). GEP pathway analysis identified DNA repair as downregulated in PTLD and EBV(+)PTLD but upregulated in EBV(-)PTLD compared IC-DLBCL. Nine DNA repair genes ( RAD51, CCNA2a, CHEK1b, POLR2Da, PCNAa, WHSC1a, GTF2H3a, ATM c, EP300 c) were differentially expressed (DE) in EBV(-)PTLD compared to IC-DLBCL (7 increased, 2 decreased, p ≤ 0.0425): where the most overexpressed gene RAD51, a critical mediator of Homologous Recombination (HR) and the Fanconi Anemia (FA) DNA repair pathways, was not DE in PTLD or EBV(+)PTLD compared to IC-DLBCL. Five a genes were increased in PTLD (p ≤ 0.0204) but not EBV(+)PTLD, 1 b was decreased in EBV(+)PTLD (p = 0.01) but not PTLD and 2 c were reduced in PTLD regardless of EBV status compared to IC-DLBCL (p ≤ 0.0024). Twenty-8 genes were DE in EBV(+)PTLD compared to IC-DLBCL. Of these, 23 were downregulated (p ≤ 0.0418) and included HR, FA and mismatch repair genes ( CHEK1, CHEK2, BRAC1/FANCS, FANCA, FANCB, FANCE, MSH2, MSH6). All but 2/23 were similarly expressed in PTLD compared to IC-DLBCL of which 11 were significant (p ≤ 0.0356). The five upregulated genes ( ZBTB32, DDB2, NBN, POLB, RBX1, p ≤ 0.0092) were also upregulated in PTLD (p ≤ 0.0042) but not EBV(-)PTLD compared to IC-DLBCL. Two additional genes ( SKP1, FBXW7) were also identified as increased in EBV(+)PTLD and PTLD (p ≤ 0.0204) compared to IC-DLBCL. In contrast, SKP1 was increased (p = 2.7E-4) and FBXW7 was decreased (p = 0.3610) in EBV(-)PTLD compared to IC-DLBCL. Together the protein products of RBX1, SKP1 and FBXW7 comprise the functional cores of a complete UPS complex, the SCF FBXW7 E3 ubiquitin ligase which have been reported to regulate the DNA damage response and promote genomic stability. Conclusions: Like HIV associated DLBCL, these results show that PTLD is more genomically stable than IC-DLBCL, and that genomic stability is further enhanced in EBV(+)PTLD which have increased expression of UPS related genes. In contrast, EBV(-)PTLD have increased expression of DNA damage repair genes, including RAD51. In summary, monomorphic DLBCL PTLD tumors have enhanced genomic stability that appears to be mediated by two distinct mechanisms stratified by EBV status. Further work is required to assess if this stratification of mechanisms is unique to PTLD or reflects a more global immune impaired EBV related signature.

A Phase Ib Study of Linperlisib Monotherapy in Patients with Relapsed or Refractory B-Cell Malignancies

Background: B-cell malignancies, a diverse and complex spectrum of lymphoproliferative disorders, are marked by a gradient of severity, ranging from indolent non-Hodgkin lymphomas (NHLs) such as follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL), to more aggressive variants of NHL like diffuse large B-cell lymphoma (DLBCL). This wide array of disorders brings unique challenges in treatment, particularly for relapsed or refractory (r/r) cases where there is a significant dearth of effective therapeutic options. The PI3K pathway, a vital regulator of cellular functions such as cell growth, survival, and differentiation, has increasingly been recognized as a promising therapeutic target in the context of B-cell lymphomas. Accumulating evidence points towards its role in the pathogenesis of these disorders, underlining the potential of PI3K inhibitors in their treatment. Linperlisib, an orally administered PI3Kδ inhibitor, has emerged as a potential candidate for the management of r/r B-cell malignancies, with a previous phase I study demonstrating its safety and suggesting preliminary clinical activity. In this light, our phase Ib study aimed to further assess the safety and efficacy of linperlisib in a broader cohort of patients with r/r B-cell malignancies. Methods: Patients with histologically or cytologically confirmed B-cell malignancies, relapsed, refractory, or lacking standard-of-care systemic therapies, were enrolled. The study encompassed several subtypes of the disease, including DLBCL, mantle cell lymphoma (MCL), FL, marginal zone lymphomas (MZL), and CLL/small-cell lymphocytic leukemia (SLL). Linperlisib was administered orally at a dose of 80 mg daily in a 28-day cycle until disease progression, onset of unacceptable toxicity, or patient withdrawal. The primary endpoint of the study was the objective response rate (ORR), as per the 2007 International Working Group Criteria for lymphomas. Secondary endpoints included safety, disease control rate (DCR), duration of response (DOR), progression-free survival (PFS), and overall survival (OS). Results: A total of 43 patients with r/r B-cell malignancies were enrolled between Jun 15, 2020, and Apr 02, 2021, and subsequently received linperlisib. At the data cutoff point of Jan 10, 2023, the median follow-up was 24.7 (range: 0.8, 30.6) months. The ORR was recorded at 46.5% (95%CI, 31.2% to 62.3%), and the DCR at 72.1% (95%CI, 56.3% to 84.7%) across all patients. More specifically, the ORR for individual diseases were as follows: 30.8% (4/13) for GCB DLBCL, 27.3% (3/11) for non-GCB DLBCL, 55.6% (5/9) for MCL, 100.0% (4/4) for FL, 66.7% (2/3) for CLL/SLL, and 66.7% (2/3) for MZL. The median DOR for the entire patient cohort was 9.3 months (95% CI: 5.4 to not reached), and the median PFS was 8.3 months (95% CI: 3.7 to not reached). The median OS was not reached, with 1-year and 2-year OS rates of 79.1% (95% CI: 63.6% to 88.5%) and 76.7% (95% CI: 61.1% to 86.8%), respectively. In terms of safety, the most common adverse events of grade 3 or higher were decreased neutrophil count, infectious pneumonia, and decreased white blood cell count (Table 1). Conclusion: In summary, our study underscores the significant potential of linperlisib in providing broad anti-tumor activity across a range of r/r B-cell malignancies, including DLBCL, MCL, MZL, FL, and SLL/CLL. These encouraging results justify further investigation of linperlisib, both as a monotherapy and in combination with other therapeutic agents. It is hoped that these findings will help evolve and enhance our treatment strategies for B-cell malignancies and ultimately improve patient outcomes.

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.

Abstract 4673: Therapeutic potential of polatuzumab vedotin for the treatment of diffuse large B-cell lymphoma

Abstract Background Diffuse large B-cell lymphoma (DLBCL) is typically treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). Although most patients can be cured with R-CHOP, up to one-third of them relapses with a dismal outcome in most cases. Numerous approaches have been attempted to improve the treatment outcomes with R-CHOP. Polatuzumab vedotin is an antibody-drug conjugate targeting CD79b, which is ubiquitously expressed on over 90% of B-cell NHL malignancies, including DLBCL. To determine the potential of polatuzumab vedotin as a therapeutic agent, we evaluate its potency across a series of DLBCL patient derived xenograft (PDX) models. Method We established and characterized a series of DLBCL lymphoma PDX models. Eight DLBCL PDX models were selected for polatuzumab vedotin efficacy study, of which three are GCB DLBCL. Gene expression analysis of these lymphoma PDX models was performed using an Illumina NovaSeq 6000 system following Illumina-provided protocols for 2 × 150 paired-end sequencing. The expression status of CD79b on these DLBCL PDX models was also evaluated by IHC. Conclusion Our data showed that a single dose of polatuzumab vedotin at just 2 mg/kg could reached a comparable effect with R-CHOP and no significant body weight loss was observed. Our result showed that there is no apparent correlation between polatuzumab vedotin responses (TGI) and the CD79b expression (IHC Score). Although polatuzumab vedotin demonstrated encouraging activity in the treatment of DLBCL, some of the PDX models are also resistant to polatuzumab vedotin treatment. To further investigate the molecular parameters of the relative sensitivity of polatuzumab vedotin. Gene expression profiling of those PDX models and their sensitivity to polatuzumab vedotin were conducted, we found that the expression level of BCL-XL was correlated with reduced sensitivity to polatuzumab vedotin. Citation Format: Hui Qi, Fuyang Wang, Bingrui Han, Xiaomin Wang, Xiangnan Qiang, Zhixiang Zhang, Qiangyang Gu. Therapeutic potential of polatuzumab vedotin for the treatment of diffuse large B-cell lymphoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4673.

Clinicopathologic features and abnormal signaling pathways in plasmablastic lymphoma: a multicenter study in China

BackgroundPlasmablastic lymphoma (PBL) is a rare but aggressive B-cell lymphoma subtype with poor prognosis. Knowledge about the etiology, clinicopathologic and molecular features, and outcomes of PBL is limited. This study aimed to examine the clinicopathologic characteristics, therapeutic approaches, and clinical outcomes of PBL patients in a Chinese population.MethodsA total of 102 PBL patients were recruited from three cancer centers. The pathologic features and clinical outcomes of 56 patients with available treatment details and follow-up data were reviewed and analyzed. RNA sequencing was performed in 6 PBL and 11 diffuse large B-cell lymphoma (DLBCL) patients.ResultsMost patients in our cohort were male (n = 36, 64.3%), and 35 patients presented with Ann Arbor stage I/II disease at diagnosis. All these patients showed negative findings for human immunodeficiency virus, and the vast majority of patients in our cohort were immunocompetent. Lymph nodes (n = 13, 23.2%) and gastrointestinal tract (n = 10, 17.9%) were the most commonly involved site at presentation. Post-treatment complete remission (CR) was the only prognostic factor affecting overall survival (OS) and progression-free survival (PFS) in the multivariate analysis. RNA-seq demonstrated that B-cell receptor (BCR), T-cell receptor (TCR), P53, calcium signaling, and Wnt signaling pathways were significantly downregulated in PBLs compared with GCB (or non-GCB) DLBCLs.ConclusionsIn this multicenter study in the Chinese population, PBL mainly occurred in immunocompetent individuals and most patients present with early-stage disease at diagnosis. Post-treatment CR was an important prognostic factor affecting OS and PFS. RNA-seq showed that the B-cell receptor (BCR), P53, calcium signaling, cell adhesion molecules, and Wnt signaling pathways significantly differed between PBL and GCB (or non-GCB) DLBCL, which provided theoretical basis for its pathogenesis and future treatment.

Correlative Biomarkers for CART19 Response in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Introduction: CD19 CAR T cell (CART19) therapy has changed the paradigms for managing relapsed diffuse large B cell lymphoma (DLBCL) however there remain challenges to achieving long term durable responses for majority of patients. Studies of lymphoma microenvironment (LME) hypothesize LME may affect CART19 response (Kotlov et al, Cancer Discov, 2021). In this study, we assessed the somatic mutation and LME profile through whole exome (WES) and RNA-sequencing (RNA-Seq) of patients treated with CART19 therapy to identify biomarkers of response. Methods: Patients from our center treated with commercial CART19 cells and biospecimen consents were identified for tumor biopsy samples pre and post-CART19 therapy. Matched normal were collected when available. WES and RNA-Seq were performed on tumor samples. Cell of origin (COO), double hit gene signatures (DHITs), and lymphoma microenvironment (LME) subtypes were calculated as described in Kotlov et al (Cancer Discov, 2021). The primary outcome endpoint was persistent response 6 months (mo) post CART19 infusion. Progression free (PFS) and overall survival (OS) was assessed by Kaplan Meier method and curves compared using log rank test. Results: WES and RNA-Seq were performed on 54 tumor samples from 49 patients with R/R DLBCL receiving CART19, either axicabtagene ciloleucel (n = 30), tisagenlecleucel (n = 18), or lisocabtagene maraleucel (n = 1). Samples were collected before (n=41) and after (n=13) CART19 therapy, and of these there are 5 patients with paired pre and post-CART samples. WES was successful in 49 samples from 44 patients, while RNA-Seq was successful in 51 samples from 47 patients. In 49 patients, overall response at 3 mo was 77.6%, (38/49) (59.2% CR, 18.4% PR), and PFS at 6 mo was 49% (24/49). Transformed DLBCL comprised 37% (18/49) of patients with overall response at 3 mo 89% (16/18) (61% CR, 28% PR), and PFS at 6 mo was 61% (11/18). We confirmed prognostic biomarkers to CAR-T therapy, such as increased lactate dehydrogenase (LDH, p = 0.01), standard uptake values (SUV, p = 0.02), and metabolic tumor volume (MTV, p = 0.02). Genomic features added further nuance into the understanding of CART19 outcomes (Figure 1). Single feature analysis revealed PIK3CA amplification was associated with improved PFS (p = 0.03), and MHCII RNA expression correlated with response to CART19 at 6 mo in GCB DLBCL (p = 0.03). Upregulation of MHCII is likely associated with the centrocyte-like phenotype, which was confirmed by a higher centrocyte B-cell associated gene signature in GCB in responders versus non-responders (p = 0.01) (Dybkær et al, JCO, 2015). Differential gene expression (DEG) analysis also revealed higher expression of MHCII genes in GCB-like responders and patients with transformed FL. In DLBCL, TGF-β pathway is inactivated via SMAD1 whose methylation suppress gene expression and inhibit chemo-sensitization. SMAD1 suppression is also associated with a depleted LME (Clozel et al, Cancer Discov, 2013; Stelling et al, Blood, 2018). High expression of SMAD1 was associated with being progression free at 6 mo in both activated B cell-like (ABC) (p = 0.003) and GCB (p = 0.06) DLBCL. The cohort was classified into favorable (germinal center-like and mesenchymal) and unfavorable (inflamed and depleted) LME subtypes (Figure 2). Favorable LME subtypes demonstrated superior OS relative to unfavorable subtypes (p = 0.02). Longitudinal analysis of one patient with paired pre- and post- CART19 tumor samples detected a potential mechanism of CAR-T relapse through B2M loss of function mutations in ABC DLBCL. Interestingly, the LME also switched from mesenchymal to depleted between the pre- and post-CART19 samples. Other paired samples will be further analyzed. Conclusion: Integrated molecular analysis of the LME and genomic alterations provides valuable information about correlative biomarkers for response to CART19 immunotherapy in DLBCL patients. With this cohort, PIK3CA amplification corresponded to improved PFS in DLBCL. We observed increased MHCII expression and centrocyte-like cells in GCB DLBCL CART19 responders, while SMAD1 was higher in ABC responders. Germinal center-like and mesenchymal LME exhibited increased OS, while inflamed and depleted LME subtypes had decreased OS. These genomic biomarkers for CART19 treatment response may help guide clinical treatment plans, minimize adverse events, or develop new therapeutic approaches. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting EGR1-Medited Metabolic Reprogramming to Overcome Ibrutinib Resistance in Aggressive B Cell Lymphomas

Targeting specific signaling pathways in malignant cells through the use of small-molecule kinase inhibitors has revolutionized the treatment of malignancies. Bruton tyrosine kinase (BTK), a key component of the B cell receptor (BCR) signaling pathway, is a promising molecular target in several types of lymphoma, including diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL). Ibrutinib, a selective BTK inhibitor, has achieved initial response rates as high as 70% in these lymphomas when used as a single agent. Acquired drug resistance, however, is significant and together with primary resistance makes ibrutinib by itself unsuitable if the goal of treatment is long-term survival. Therefore, understanding and targeting ibrutinib resistance mechanisms is an unmet clinical need. DLBCL, which represents 30% to 40% of newly diagnosed lymphomas, comprises two main molecular subtypes: activated B cell-like (ABC) and germinal center B cell-like (GCB). The BCR/BTK signaling pathway is essential for the growth of ABC DLBCL, which is more aggressive and less curable than GCB DLBCL with current regimens. We established two representative ABC DLBCL cell lines (TMD8 and OCI-Ly10) with ibrutinib resistance by gradually increasing the concentration of ibrutinib during passage in culture. RNA-seq analysis demonstrated that the BCR pathway gene signature is enriched in resistant cell lines when compared to parental cells. The most upregulated gene is EGR1, a transcription factor that activates multiple oncogenic pathways including MYC and E2F. Elevated EGR1 expression is also observed in ibrutinib-resistant primary mantle cell lymphoma cells when treated with ibrutinib. Using multiple metabolic and genetic approaches, we discovered that overexpression of EGR1 causes metabolic reprogramming to oxidative phosphorylation (OXPHOS) and ibrutinib resistance. Mechanistically, EGR1 mediates metabolic reprogramming through transcriptional activation of PDP1, a phosphatase that dephosphorylates and activates the E1 component of the large pyruvate dehydrogenase complex. Therefore, EGR1-mediated PDP1 activation accelerates intracellular ATP production via the mitochondrial tricarboxylic acid (TCA) cycle, leading to sufficient energy to enhance the proliferation and survival of ibrutinib-resistant lymphoma cells. Finally, we demonstrate that targeting OXPHOS with IM156, a newly developed OXPHOS inhibitor, inhibits the growth of ibrutinib-resistant lymphoma cells both in vitro and in patient-derived xenograft mouse models. These findings suggest that targeting EGR1-medited metabolic reprogramming to OXPHOS with IM156 provides a potential therapeutic strategy to overcome ibrutinib resistance in relapsed/refractory DLBCL or MCL.

BCL6 Rearranged Indolent B-Cell Lymphomas Show Mutational Profile Similar to Marginal Zone Lymphomas

Introduction Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma. It is a neoplasm of germinal center (GC) B cells with hallmark BCL2 rearrangements (BCL2-R). A distinct subgroup of indolent B cell lymphomas (iBCL), histologically revealing a follicular growth pattern but lacking BCL2-R and with BCL6 rearrangements (BCL6-R) has been described. Historically they have been classified as FL and included under the FL entity in the WHO classification. However, recent studies on diffuse Large B cell lymphomas (DLBCL) (1, 2) have described BCL6-R in ~20% of DLBCL, with a subset of cases showing concurrent gene mutations commonly found in marginal zone lymphomas (MZL) rather than those characteristic of FL or GCB DLBCL. Systematic studies elucidating the clinicopathologic and genetic features of the BCL6-R iBCL are currently lacking thus generating controversy over their appropriate classification and management. Methods The MSKCC cytogenetic lab database was searched from 01/2006 to 07/2021 and a cohort of 131 iBCL with BCL6-R (71 with BCL6-R only and 60 with both BCL6-R & BCL2-R) was obtained (Table 1). The clinical, morphologic, immunophenotypic, and molecular results (50 cases had molecular data) were reviewed. These were compared to 70 BCL2-R conventional FL (cFL; all having molecular data) and 54 BCL2-R and BCL6-R negative iBCL (17 had molecular data). ResultsBCL6-R iBCL showed distinct histological, immunophenotypic, genetic and clinical features. They showed a significantly higher number of Grade 3 FL diagnosis and CD10 negative phenotype. The most commonly mutated genes in BCL6-R only group were KMT2D (48%) and TNFAIP3 (40%); in the BCL6-R & BCL2-R group, KMT2D (72%) and BCL2 (56%); and in the cFL group KMT2D (83%), BCL2 (81%) and CREBBP (66%). Unsupervised clustering (K-Means) performed to cluster the samples based on the mutational status of about 50 genes revealed that majority of the BCL6-R only cases clustered separately from cFLs. They clustered with majority of BCL2-R and BCL6-R negative cases and revealed frequent mutations in TNFAIP3, BCL10, B2M, NOTCH2, SPEN (Fig 1). Majority (2/3) of the BCL6-R & BCL2-R cases clustered with cFL and showed mutations commonly seen in FL (Fig 1). The overall survival (OS) was significantly inferior in the BCL6-R cases (BCL6-R only as well as both BCL6-R & BCL2-R cases) in comparison to cFL even though progression free survival was shortest for cFL. ConclusionBCL6-R only iBCL are biologically distinct, cluster separately from cFL based on mutational profile, and demonstrate an MZL-like mutational profile similar to that is described for the BN2 group of DLBCL (1). These findings suggest that they are a distinct entity, different from cFL and possibly precursor lesions for BN2 DLBCL. In contrast, majority of the BCL6-R & BCL2-R cases cluster with cFL and show mutations commonly seen in FL. Finally, presence of BCL6-R was associated with a worse OS.

B-Cell Receptor Signaling Modulates Cholesterol Biosynthesis in Diffuse Large B-Cell Lymphoma

Cholesterol levels are associated with cancer progression and targeting cholesterol biosynthesis has been proposed as a therapeutic strategy in some neoplasms. It has been shown that inhibition of cholesterol synthesis in combination with chemotherapy resulted in encouraging clinical responses in patients with hematopoietic neoplasms including diffuse large B cell lymphoma (DLBCL) (Blood 2007; 109:2999-3006; Leuk. Lymphoma 2022; 63:1302-1313). Seminal work in DLBCL has shown that blocking the expression of SYK kinase, downstream of the B-cell receptor (BCR), impacted cholesterol biosynthesis (Cancer Cell 2013; 23:826-88). Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors in the endoplasmic reticulum, play a central role in lipid metabolism. Two genes, SREBF1, and SREBF2 codify three proteins (SREBP1a, SREBP1c, and SREBP2). SREBP1a and SREBP1c primarily regulate fatty acid metabolism, whereas SREBP2 is a master regulator of cholesterol biosynthesis. The underlying question of how BCR signaling is linked to the cholesterol biosynthesis encircling SREBP2 is not well understood. Using publicly available NCBI's Gene Expression Omnibus (GEO) DLBCL databases, we first analyzed the expression of genes directly regulated by SREBF2 involved in the biosynthesis of cholesterol, including ACAT1-2, HMGCS, HMGCR, MVK, PVMK, IDI1, SQLE, LSS, EBP, DHCR7, DHCR24, and NSDHL in DLBCL. We observed that SREBF2-mediated genes were upregulated in DLBCL as compared to non-neoplastic germinal center (GC) B-cells or low-grade B-cell lymphomas. These data were further confirmed by HTseq analysis of 66 DLBCL PDXs tumors established from DLBCL patients in our institution. Also, protein expression of nuclear SREBP2 was analyzed using immunohistochemistry in a DLBCL tissue microarray (n=84). Nuclear SREBP2 expression serves as an active form of transcriptional factor following cleavage. We confirmed strong nuclear positivity of SREBP2 in all non-GCB DLBCL; however, a subset of GCB DLBCL exhibited moderate nuclear SREBP2 positivity as compared to low-grade follicular lymphomas or reactive lymph node (control). These data suggest that SERBP2 is a key element in cholesterol biosynthesis, specifically upregulated, in non-GCB DLBCL. Next, in-vitro functional studies were performed to connect BCR signaling with SERBP2. We either genetically knock out the expression of IgH using CRISPR/Cas9 or pharmacologically inhibited Src kinase (downstream of the BCR signaling) with Dasatinib in non-GCB DLBCL cell lines. Both approaches reduce SREBP2 expression and its cholesterol-related downstream genes. In contrast, recovering IgH expression rescues the SREBP2-mediated cholesterol biosynthesis pathway establishing a direct role of BCR signaling in the regulation of the cholesterol pathway in non-GCB DLBCL through SREBP2. Mechanistically using Reverse Phase Protein Array (RPPA) proteomic analysis, we have identified cyclin-dependent kinase 1 (CDK1), a key player in cell cycle regulation, as a potential kinase involved in the phosphorylation of SREBP2 by activated BCR signaling. Our results suggest that CDK1 interacts with SREBP2 (as per colocalization studies), and that CDK1-mediated phosphorylation of SREBP2 preserves its stability to support enhanced cholesterol synthesis in non-GCB DLBCL cell lines. Although directly inhibiting of SREBPs is challenging, a promising approach is to inhibit SREBP translocation from the ER to the Golgi using PF-429242. Decreasing expression of SREBP2 at the genetic level or blocking the processing of SREBP2 with PF-429242 strongly reduces cell viability of DLBCL cell lines, with non-GCB DLBCL cell lines being more sensitive (IC50= 60-225nM) than the GCB DLBCL cells (IC50=200-330 nM). We also observed, in vitro, that co-treatments with PF-429242 and doxorubicin resulted in a synergistic response to doxorubicin in DLBCL cell lines of GCB, non-GCB and "double hit" suggesting that inhibiting SREBP2 could be a potential adjuvant therapeutic approach for DLBCL patients. In summary, cholesterol biosynthesis is tightly regulated by BCR signaling through SREBP2, specifically in non-GCB DLBCL. SREBP2 is a potential promising adjuvant therapeutic target for DLBCL as PF-429242 enhances responses to doxorubicin in cell lines expanding the therapeutic window of doxorubicin by increasing its efficacy.

Mivavotinib, a Syk Inhibitor, in Relapsed/Refractory (R/R) Non-GCB Diffuse Large B-Cell Lymphoma (DLBCL) with or without MYD88 and/or CD79 Mutations: A Phase 2 Study

Background: Spleen tyrosine kinase (Syk) is a key signaling protein that activates survival pathways in B-cell lymphomas. The non-germinal center B-cell like (non-GCB) or activated B-cell like (ABC) cell-of-origin subtype of DLBCL has greater reliance on the B-cell receptor pathway and Syk signaling for survival, compared with the GCB subtype. Multiple signaling pathways are activated by Syk in ABC DLBCL (NF-κB and phosphoinositide 3-kinase [PI3K]), whereas only the PI3K pathway is activated by Syk in GCB DLBCL. MYD88 and/or CD79b mutations are strongly enriched within ABC DLBCL and are associated with poor prognosis. Syk hyperactivation has been observed in ABC DLBCL cell lines harboring MYD88/CD79 mutations, with activation of NF-κB via Syk-mediated signaling through both the BCR and the toll-like-receptor (TLR)-MYD88 pathways. Mivavotinib is an orally dosed, potent, and selective Syk inhibitor, which showed potent single agent activity in preclinical studies in ABC and GCB DLBCL cell lines, with the strongest activity against those harboring MYD88 and/or CD79b mutations. In the clinical setting, heavily pre-treated all-comer R/R DLBCL patients receiving single agent mivavotinib at the recommended phase 2 dose (100 mg once daily [QD]) showed an overall response rate (ORR) of 33% (16% complete responses [CR]) with durable responses (median duration of response [DoR] not reached [95% CI: 7 months - NE]) and a favorable safety profile. Subgroups by cell-of-origin showed a substantially higher ORR in patients with the non-GCB subtype (53%; 95% CI: 27, 79) than GCB (22%; 95% CI: 0.11, 0.37), and a median DoR of 15.7 months (95% CI: 2.2, NE) in non-GCB responders. CX-659-401 (NCT05319028) is an ongoing study evaluating two dosing regimens of mivavotinib monotherapy in patients with R/R non-GCB DLBCL, in whom circulating tumor DNA-based next generation sequencing (NGS) will be used to determine MYD88/CD79b mutational status. The goals of this strategy are to confirm the activity of mivavotinib previously observed in non-GCB DLBCL, evaluate its activity in DLBCL according to mutational status of MyD88/CD79b, and optimize the dose and schedule in this biomarker-defined patient population. Methods: This multicenter, open-label, phase 2 study will enroll patients with histologically confirmed de novo or transformed DLBCL that is non-GCB by immunohistochemistry (IHC) per Hans algorithm or ABC by gene expression profiling, including primary mediastinal and primary extranodal DLBCL. Patients must have had at least 2 but no more than 5 prior lines of systemic anticancer therapy. Following standard first-line R-CHOP (or equivalent), patients must have received 2nd-line salvage therapy with or without autologous stem cell transplant (ASCT), and/or CAR-T therapy, unless ineligible for 2nd-line salvage, ASCT, or CAR-T. Additional key eligibility criteria include age >18 years, ECOG performance status 0-2, and FDG-PET-avid measurable disease per 2014 Lugano criteria. DLBCL patients with primary brain or secondary central nervous system involvement with active disease will be excluded. Patients will be randomized to 1 of 2 arms (n=25 planned per arm): (1) 100 mg QD; or (2) 120 mg QD for 2 weeks followed by 80 mg QD thereafter; in 28-day cycles until disease progression or unacceptable toxicity. The primary endpoints are ORR (by independent radiology review according to 2014 Lugano Criteria) and safety/tolerability. Secondary endpoints include DoR, progression-free survival (PFS), overall survival (OS), and CR rate. Exploratory endpoints include pharmacokinetic, biomarker assessments, and investigator-assessed ORR, DoR, PFS, and CR rate. Results of this study will help to define the optimal dose and provide confirmatory efficacy and safety data of mivavotinib in patients with non-GCB DLBCL, including MYD88/CD79b-mutated DLBCL to guide further expansion in these biomarker-defined cohorts.

Single-Cell Multi-Omic and Spatial Analysis of Nodal B Cell Non-Hodgkin Lymphomas Reveals Plasticity in B Cell Maturation As a Driver of Intratumor Heterogeneity

Introduction Intratumor heterogeneity (ITH) is increasingly recognized as an important factor in the variability of B cell non-Hodgkin lymphoma (B-NHL) patient outcomes. It is well established that distinct nodal B cell non-Hodgkin lymphoma (NBNHL) entities originate from different stages of the humoral affinity maturation process, but drivers of ITH are not well understood. In this study, we aimed to investigate whether the maturation process is a driver of ITH. Methods 51 diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and non-malignant reactive lymph node (rLN) samples from distinct patients were sequenced with CITE-Seq (coupling single-cell RNA-seq with surface epitope profiling), highly multiplexed immunohistochemistry (IHC) with 57 proteins, and single-cell B cell receptor sequencing. Transcription factor activites were inferred from gene expression using the SCENIC package. (Fig 1a)Results Using the rLN samples (n = 8), a single-cell reference map of nodal B cell maturation states was constructed based on known B cell maturation states and their markers. This reference map was used to classify maturation states in malignant samples, which were verified and refined against their maturation marker profiles. Comparable to the non-malignant context in rLN samples, variability in maturation states was also observed for malignant cells in most NBNHL tumors. Although commonalities in composition existed between samples within entities, such as a dominant pre-GC mantle cell population in MCL and abundance of memory B cells in MZL, we observed high variability in maturation state proportions between these samples (Fig 1b). Several GCB and ABC DLBCL samples contained subpopulations of both GC and post-GC phenotypes. BCR-sequencing confirmed that different intra-tumor maturation states observed were monoclonal, and therefore arose from the same cell of origin. Common epigenetic drivers of the maturation processes, such as the transcription factors EZH2 and EGR1, were upregulated in subpopulations advanced in maturation. Known pathways common driving malignant transformation in NBNHL pathogenesis, BCL6 and BCL2, were upregulated in centroblasts and post-GC subpopulations respectively in tumors of the same entities. Conclusions Our findings reveal maturation is not halted during malignancy, but rather remains a plastic and divergent process. Ongoing maturation and malignant transformation processes are intertwined as the driving forces of intratumor heterogeneity, blurring the known boundaries of NBNHL entities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract A05: Olfactory receptors as a new therapeutic target in lymphomas

Abstract Background. Diffuse large B cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are aggressive lymphomas in which up to 40% of patients are still in need of therapy. G protein-coupled receptors (GPCRs) are integral membrane proteins that mediate cellular responses to endogenous and exogenous stimuli and are implicated in the pathophysiology of various diseases: CXCR3/4/5, S1PR1/2/3, or GPR34 are GPCRs already studied in lymphomas. The olfactory receptors (ORs) are also GPCRs, initially identified and characterized for their expression in olfactory cells but now shown to be expressed in various tissues. OR51E1 is known as prostate-specific G protein-coupled receptor 2 (PSGR2) due to its expression in prostate cancer, OR2B6 and OR2W3 are expressed in breast cancer, and OR51E1 in melanoma. Here, we studied OR13A1 in lymphoma cell lines and clinical specimens. Methods. RNA-Seq and real-time PCR for evaluating the expression of ORs in lymphoma cell lines. Gene expression data for clinical samples were obtained from public datasets. OR13A1 silencing was performed with both siRNAs (OCI-Ly1 and U2932) and doxycycline-inducible shRNAs (U2932 and VAL); viability was assessed after 3 days using MTT and even longer with live imaging. Computer modelling was performed using available ligands for the murine orthologue Olfr211. Results. Various ORs were expressed across a large panel of B cell lymphoma cell lines. In particular, OR13A1 was expressed in 35/47 cell lines and, mostly, derived from DLBCL and MCL. Among DLBCL, the expression was higher in cell lines derived from the germinal center (GC) B-cell type (GCB) rather than from activated B cell like (ABC) DLBCL (P value = 0.03). A similar pattern was observed in DLBCL patients: higher expression in GCB than ABC and in EZB than MCD (P < 0.001). OR13A1 also appeared higher in normal GC cells than in naïve and memory B cells. OR13A1 expression appears to identify GCB DLBCL (P value = 0.009) and MCL (P value = 0.006) patients with inferior outcome. Silencing of OR13A1 with siRNAs in GCB and ABC DLBCL cells (OCI-Ly1 and U2932) caused > 50% reduction in cell viability, and downregulation of pERK levels. Viability data were confirmed using a doxycycline-inducible shRNA system and an Incucyte live cell imaging instrument. Natural compounds reported as ligands for the murine orthologue Olfr211 or in silico identified to bind as agonists (ex: tyramine) increased cell line growth and compounds predicted to bind as antagonists (ex: cyclohexanone) decreased the cell viability in the presence but not in the absence of the GPCR. Conclusion. OR13A1 gene appeared more abundantly expressed in lymphomas compared to others ORs, in particular it was more expressed in MCL and in GCB DLBCL with a possible impact on clinical outcome. Genetic and chemical experiments showed that this GPCR has an essential function in lymphoma cells and represents a novel therapeutic target. Citation Format: Giulio Sartori, Luciano Cascione, Milos Matkovic, Hiro Matsunami, Andrea Cavalli, Francesco Bertoni. Olfactory receptors as a new therapeutic target in lymphomas [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A05.