Subtype Of Diffuse Large B-cell Lymphoma Research Articles

Abstract 2265: Distinct alternative splicing patterns in genetic subtypes of diffuse large B-cell lymphoma

Abstract Advances in sequencing techniques have made molecular subgrouping of Diffuse Large B-cell Lymphoma (DLBCL) based on DNA cahnges, which has led to the development of Lymphgen classification. By the way, although alternative splicing (AS) in cancer cells contributes to proliferation, invasion and metastasis, and immune escape, their role and association with DLBCL molecular subtypes are unknown. Therefore, we analyzed whole-exome sequencing (WES) and bulk RNA-Seq data paired in the public EGA dataset (EGAD00001003600) to explore the patterns of AS events that differ between Lymphgene subtypes. The WES data of 620 DLBCL samples that passed quality control were aligned to the reference using BWA-MEM, and variants were called with Mutect2. We used the information of copy number variants provided by cBioPortal. The RNA-Seq analysis was performed using the STAR-RSEM pipeline and differential splicing events were detected using rMATS to compare subtypes. Among 620 DLBCL samples, there were 19 MCDs, 95 EZBs, 22 BN2s, and 137 ST2s (50 samples with multiple genetic classes, and 291 samples unclassified). Mutations in spliceosome complex genes (ZRSR2 and SF1) were found in 14 and 27 samples, respectively. In BN2 and MCD subtypes, splicing events with significantly different exon inclusion levels were observed at a high frequency of 29 and 16 occurrences, respectively, compared to other subtypes. (P-value < 0.05). In contrast, retained intron is the only event observed in the EZB subtype (Table 1). Samples with mutations in ZRSR2 and SF1 showed four and two aberrant events, respectively, compared to samples without these mutations. Of note, rMATS detected an alternative 3’ splice site event in exon 6 of CD37 in the MCD. CD37 is involved in tumorigenesis in leukocytes and has been suggested as a potential biomarker in blood cancer, and its dysregulation may have influenced the poor prognosis of MCD. In summary, molecular subtypes of DLBCL had unique features in terms of alternative splicing. Functional consequences of these selective exon usages for each DLBCL subtype are being warranted. Citation Format: Sanyeowool An, Youngil Koh, Sung-Soo Yoon. Distinct alternative splicing patterns in genetic subtypes of 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 2265.

Artesunate induces ferroptosis by regulating MT1G and has an additive effect with doxorubicin in diffuse large B-cell lymphoma cells

Diffuse Large B-cell lymphoma (DLBCL) is a highly aggressive disease with heterogeneous outcomes and marked variability in the response to chemotherapy. DLBCL comprises two major subtypes: germinal centre B-cell-like (GCB) and activated B-cell-like (ABC). Our study highlights the extensive antitumour activity of artesunate (ART) against both major DLBCL subtypes. Transcriptome analysis suggests the potential involvement of ferroptosis in artesunate-induced cell death. Because of low glutathione (GSH) and glutathione peroxidase 4 (GPX4) levels, along with the accumulation of free iron (Fe2+), artesunate induces the excessive production of reactive oxygen species (ROS), ultimately leading to ferroptosis, a form of cell death driven by phospholipid peroxidation. A putative target of artesunate, metallothionein 1G (MT1G), was selected for further analysis. Subsequent studies revealed that inhibiting MT1G expression in vitro significantly impedes the ferroptosis-promoting activity of artesunate by reducing lipid peroxidation and iron accumulation. We also showed that the combination of artesunate and doxorubicin had a marked additive inhibitory effect on GCB and ABC DLBCL cells. In conclusion, artesunate induces ferroptotic death in GCB and ABC DLBCL cells by attenuating the GPX4/GSH antioxidant defence system and increasing intracellular iron levels, indicating its therapeutic potential for relapsed or refractory DLBCL.

Epstein-Barr virus-positive iris diffuse large B-cell lymphoma detected by metagenomic next-generation sequencing

PurposeEpstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) is a relatively rare subtype of DLBCL. Herein, we report a case of a patient with EBV-positive iris DLBCL after undergoing penetrating keratoplasty and discuss its possible pathogenesis.MethodsA 72-year-old male patient presented to our hospital with progressive blurring of vision in the left eye for the past 4 months. Small white nodular lesions were observed on the iris and retinal surface of the left eye, with a white cloud-like opacity in the vitreous cavity.ResultsThe patient was eventually diagnosed with EBV-positive iris DLBCL after undergoing pathological and metagenomic tests. After injecting methotrexate in the left vitreous cavity and administering systemic and local antiviral treatments, the ocular lesions disappeared.ConclusionEBV infection, drug immunosuppression, and aging-related immune deterioration may play significant roles in the pathogenesis of EBV-positive iris DLBCL.SynopsisEpstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) is a new subtype of DLBCL, which rarely occurs. Herein, we report a case of a patient with EBV-positive iris DLBCL after undergoing penetrating keratoplasty and discuss its possible pathogenesis.

Epiregulin: A new prognostic molecule in non-Hodgkin lymphoma.

Epiregulin is a molecule that plays a role in cell proliferation, tumor development, inflammation, and angiogenesis in malignant diseases. Our study aims to reveal, for the first time, the predictive value of this molecule in non-Hodgkin lymphoma (NHL) and its association with disease stage, cell type, and extranodal involvement. The study is an observational case-control trial involving 60 newly diagnosed NHL patients and 60 healthy individuals (control group) between 18 and 75years old. Demographic characteristics of all volunteers, stages of patients' illnesses and lymphoma cell types, hemogram, biochemistry tests, beta 2-microglobulin, C-reactive protein (CRP), and epiregulin levels were measured and statistically evaluated. Epiregulin levels were significantly higher in NHL patients compared to the control group (P < 0.0001). Similarly, a significant increase in epiregulin levels was observed in advanced NHL patients. Furthermore, the most common NHL subgroup within the NHL group, diffuse Large B Cell Lymphoma (DLBCL), and the subgroup with extranodal involvement also had significantly higher levels of epiregulin. A positive correlation was found between the epiregulin molecule and CRP, beta 2-microglobulin, and lactate dehydrogenase (LDH) levels in NHL patients. Our study suggests that serum epiregulin levels, discovered to increase in NHL patients for the first time, may be an independent predictive molecule in an advanced stage, extranodal involvement, and the DLBCL subtype of this disease. Epiregulin positively correlates with prognostic molecules such as beta 2-microglobulin, LDH, and CRP. Illuminating its potential role in NHL pathogenesis could make epiregulin a vital drug target for treatment.

Response to Bruton’s tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy

Diffuse large B cell lymphoma (DLBCL) is an aggressive, profoundly heterogeneous cancer, presenting a challenge for precision medicine. Bruton's tyrosine kinase (BTK) inhibitors block B cell receptor (BCR) signaling and are particularly effective in certain molecular subtypes of DLBCL that rely on chronic active BCR signaling to promote oncogenic NF-κB. The MCD genetic subtype, which often acquires mutations in the BCR subunit, CD79B, and in the innate immune adapter, MYD88L265P, typically resists chemotherapy but responds exceptionally to BTK inhibitors. However, the underlying mechanisms of response to BTK inhibitors are poorly understood. Herein, we find a non-canonical form of chronic selective autophagy in MCD DLBCL that targets ubiquitinated MYD88L265P for degradation in a TBK1-dependent manner. MCD tumors acquire genetic and epigenetic alterations that attenuate this autophagic tumor suppressive pathway. In contrast, BTK inhibitors promote autophagic degradation of MYD88L265P, thus explaining their exceptional clinical benefit in MCD DLBCL.

An Assessment of Primary Cardiac Lymphoma: A Rare Case in Indonesia

Introduction: : Primary cardiac lymphoma (PCL) is an uncommon malignancy with a high mortality rate. The disease is commonly found as non-Hodgkin’s lymphoma (NHL), mainly located in the heart or pericardium. PCL is difficult to diagnose due to its unspecific clinical manifestations. Echocardiography, computed tomography (CT) scanning, and magnetic resonance imaging (MRI) can help in diagnosing PCL, but histopathological examination is the gold standard for a definitive diagnosis. Therefore, this study aimed to compare the case of malignant cardiac lymphoma and MRI with earlier related literature. Case Presentation: This study reported a case of PCL in 52 years old male who came to Cipto Mangunkusumo Hospital with intra cardiac tumour and underwent surgery with median sternotomy. CT scan of the chest showed a mass in the left atrial and no significant abnormalities in other organs. Histopathological examination showed the morphologic feature of diffuse monotonous proliferation of atypical large B lymphocytes. The lymphoma diagnosis was supported by immunohistochemistry including CD20, MUM1, and Ki67 positivity. Conclusions: PCL was difficult to diagnose due to nonspecific clinical manifestations. Immunostaining helped to diagnose more specifically and determine the treatment plan. Histologic finding from PCL was mostly Diffuse Large B-cell lymphoma (DLBCL) and the prognosis was poor. Therefore, identifying the subtype of DLBCL was important for prognostic.

Ibrutinib Plus RCHOP versus RCHOP Only in Young Patients with Activated B-Cell-like Diffuse Large B-Cell Lymphoma (ABC-DLBCL): A Cost-Effectiveness Analysis.

The standard treatment for Diffuse Large B-Cell Lymphoma (DLBCL) is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (RCHOP). However, many patients require subsequent treatment after relapsed disease. The ABC subtype of DLBCL (ABC-DLBCL) has a worse prognosis, and the PHOENIX trial explored adding ibrutinib to RCHOP for this patient population. The trial showed favorable outcomes for younger patients, and our study aimed to inform clinical decision-making via a cost-effectiveness model to compare RCHOP with and without ibrutinib (I-RCHOP). A Markov decision analysis model was designed to compare the treatments for patients younger than 60 years with ABC-DLBCL. The model considered treatment pathways, adverse events, relapses, and death, incorporating data on salvage treatments and novel therapies. The results indicated that I-RCHOP was more cost-effective, with greater quality-adjusted life years (QALY, 15.48 years vs. 14.25 years) and an incremental cost-effectiveness ratio (ICER) of CAD 34,111.45/QALY compared to RCHOP only. Sensitivity analyses confirmed the model's robustness. Considering the high market price for ibrutinib, I-RCHOP may be more costly. However, it is suggested as the preferred cost-effective strategy for younger patients due to its benefits in adverse events, overall survival, and quality of life. The decision analytic model provided relevant and robust results to inform clinical decision-making.

CD5 Gene Signature Identifies Diffuse Large B-Cell Lymphomas Sensitive to Bruton's Tyrosine Kinase Inhibition.

A genetic classifier termed LymphGen accurately identifies diffuse large B-cell lymphoma (DLBCL) subtypes vulnerable to Bruton's tyrosine kinase inhibitors (BTKis), but is challenging to implement in the clinic and fails to capture all DLBCLs that benefit from BTKi-based therapy. Here, we developed a novel CD5 gene expression signature as a biomarker of response to BTKi-based therapy in DLBCL. CD5 immunohistochemistry (IHC) was performed on 404 DLBCLs to identify CD5 IHC+ and CD5 IHC- cases, which were subsequently characterized at the molecular level through mutational and transcriptional analyses. A 60-gene CD5 gene expression signature (CD5sig) was constructed using genes differentially expressed between CD5 IHC+ and CD5 IHC- non-germinal center B-cell-like (non-GCB DLBCL) DLBCLs. This CD5sig was applied to external DLBCL data sets, including pretreatment biopsies from patients enrolled in the PHOENIX study (n = 584) to define the extent to which the CD5sig could identify non-GCB DLBCLs that benefited from the addition of ibrutinib to rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). CD5 expression was observed in 12% of non-GCB DLBCLs. CD5+ DLBCLs displayed transcriptional features of B-cell receptor (BCR) activation and were enriched for BCR-activating mutations known to correlate with BTKi sensitivity. However, most CD5+ DLBCLs lacked canonical BCR-activating mutations or were LymphGen-unclassifiable (LymphGen-Other). The CD5sig recapitulated these findings in multiple independent data sets, indicating its utility in identifying DLBCLs with genetic and nongenetic bases for BCR dependence. Supporting this notion, CD5sig+ DLBCLs derived a selective survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study, independent of LymphGen classification. CD5sig is a useful biomarker to identify DLBCLs vulnerable to BTKi-based therapies and complements current biomarker approaches by identifying DLBCLs with genetic and nongenetic bases for BTKi sensitivity.

Abstract C053: Multi-omics guided design of lymphoid organoids to study cooperative cell signaling in lymphomas

Abstract Our collaborative research (Cell1) used 1001 patients of diffuse large B-cell lymphoma (DLBCL) to show that genetic alterations interact with clinical factors to impact overall survival (OS) in DLBCL. B cell receptor (BCR) signaling-driven activated B cell-like (ABC) DLBCL subtypes is the most chemo-resistant subtype to the frontline therapy R-CHOP, with 40% of patients experiencing no response or relapse. We have systematically examined disparities in lymphoma outcomes and identified African-American (AA) patients as a poor-risk population in DLBCL, with a diagnosis age 10 years younger than other racial groups. We found that AA-DLBCL patients experienced inferior 5-year OS compared to non-Hispanic whites (38% vs 46%)2,3. We performed the first-ever characterization of genetic alterations among AA-DLBCL patients and demonstrated distinct mutation patterns across DLBCL arising in AA ancestry groups4. While some of the most frequent genetic mutations in AA-DLBCL are related to chromatin and epigenetics, few studies have investigated racial disparities in patients with aggressive ABC DLBCLs. The mechanisms that determine this racial inequality are unknown but may lie in the particular spectrum of mutations that act in concert with intricate survival signals imparted by the lymph node tumor microenvironment (Ly-TME). The pathogenesis of ABC DLBCLs has been linked to constitutive activation of and cooperation between the BCR and Toll-like receptor (TLR) pathways. These pathways are emerging as novel therapeutic targets, with a particular interest in MALT1 (mucosa-associated lymphoid tissue translocation protein 1). However, the genetic heterogeneity of ABC DLBCLs and the cooperative signaling mediated by the Ly-TME have led to discrepancies in drug responses. As a first step towards understanding Ly-TME in DLBCLs (Nature Materials5), we used DLBCL patient samples from multiple cohorts, covering &amp;gt;1100 samples and imaging analysis to determine immune and ECM cues in the lymphoid TME. These samples were not classified into racial categories. We designed a biomaterials-based modular lymphoma organoid that presents specific integrin ligands at controlled densities to DLBCLs. Human ABC-DLBCLs responded to MALT1 inhibition in an integrin-ligand-dependent manner. TLR activation with TLR agonists rescued cells from MALT1 inhibitors, possibly due to increased MALT1 expression. The inclusion of T-cell signals increased MALT1 expression and rescued cells from MALT1 inhibitor-mediated killing in cell lines and human PDX organoids. Changes in RNA-seq and phosphorylation in the TLR &amp; BCR pathways were used to inform effective combination therapy approaches with MALT1 inhibitors in vitro and in vivo5. Ongoing work is determining the racial disparities in AA lymphomas through complex interactions between Ly-TME and DLBCLs. 

M6A-related lncRNAs as potential biomarkers and the lncRNA ELFN1-AS1/miR-182-5p/BCL-2 regulatory axis in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid subtype. However, unsatisfactory survival outcomes remain a major challenge, and the underlying mechanisms are poorly understood. N6-methyladenosine (m6A), the most common internal modification of eukaryotic mRNA, participates in cancer pathogenesis. In this study, m6A-associated long non-coding RNAs (lncRNA) were retrieved from publicly available databases. Univariate, LASSO, and multivariate Cox regression analyses were performed to establish an m6A-associated lncRNA model specific to DLBCL. Kaplan-Meier curves, principal component analysis, functional enrichment analyses and nomographs were used to study the risk model. The underlying clinicopathological characteristics and drug sensitivity predictions against the model were identified. Risk modelling based on the three m6A-associated lncRNAs was an independent prognostic factor. By regrouping patients using our model-based method, we could differentiate patients more accurately for their response to immunotherapy. In addition, prospective compounds that can target DLBCL subtypes have been identified. The m6A-associated lncRNA risk-scoring model developed herein holds implications for DLBCL prognosis and clinical response prediction to immunotherapy. In addition, we used bioinformatic tools to identify and verify the ceRNA of the m6A-associated lncRNA ELFN1-AS1/miR-182-5p/BCL-2 regulatory axis. ELFN1-AS1 was highly expressed in DLBCL and DLBCL cell lines. ELFN1-AS1 inhibition significantly reduced the proliferation of DLBCL cells and promoted apoptosis. ABT-263 inhibits proliferation and promotes apoptosis in DLBCL cells. Invitro and invivo studies have shown that ABT-263 combined with si-ELFN1-AS1 can inhibit DLBCL progression.

Raman Spectroscopy Imaging As a Tool to Classify Diffused Large B-Cell Lymphoma Subtypes

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a clinically heterogenous, aggressive disease. The ‘cell-of-origin’ classification (COO) distinguishes two major DLBCL subtypes, GCB (germinal center B cell-like) and ABC (activated B cell-like) that are derived from distinct putative cell of origin, differ in prognosis and transcriptional pattern. Comprehensive cluster classification (CCC) based on unbiased “consensus clustering” of transcriptomic microarray data has led to the identification of subtypes that differ in energy source utilization and bioenergetic profiles The oxPhos DLBCL subtype, unlike non-oxPhos, is characterized by increased mitochondrial oxidative phosphorylation and energy transduction. Although COO class assignment can be achieved with multiple clinical diagnostic tools (including transcript-based or immunohistochemical approaches), there is no clinical diagnostic tool appropriate for COO classification. In Raman effect, laser light, as a result of internal oscillations of molecules, is scattered with a change in wavelength, giving a spectrum characteristic for a given substance. This allows the chemical composition of mixtures and even cells to be determined on the basis of the obtained spectra. Thus, it also allows to determine differences in metabolism, including energy metabolism of lymphoma cells. The aim of the study was to use the RS images to recognize and classify DLBCL cells. MATERIALS AND METHODS: DLBCL cell lines (OCI-Ly4, Pfeiffer, Toledo, DHL4, DHL6 , OCI-Ly1 and OCI-Ly7) with known COO and CCC subtype assignments, and B cells isolated from the blood of healthy donors were used. For Raman analysis cells were fixed in 0.5% glutaraldehyde and imaged at 533 and 633 nm emission wavelengths under a WITec Raman microscope. The collected spectral images were subjected to chemometric analyses: characteristic bands analysis, k-means cluster analysis (KMCA), principal component analysis (PCA) and least squares discriminant analysis (PLS-DA) using dedicated software. RESULTS: The analysis of Raman spectra revealed clear changes in the structure and composition of cells after neoplastic transformation. Chemometric analysis of spectral bands showed greater proportion of proteins (1008, 1041 and 1660 cm-1) and lipids (1440, 1240 and 2850 cm-1) in DLBCL cells then B cells from healthy donors. More nucleic acids were observed (733, 790 , 1583 cm-1) in normal B cells that at least partially resulted from the greater ratio of the nucleus to the cytoplasm. The differences resulted in clear separation of malignant and healthy cells in PCA and PLS-DA. Similar approach allowed to characterize differences between ABC vs GCB subtype cell lines and oxPhos vs non-oxPhos subtype. Based on the specific differences between healthy B cells and DLBCL cells a predictive model was built using PLS-DA that accurately determines the chance of the analyzed cell, first, being a lymphoma cell, and second, belonging to the ABC vs GCB or oxPhos vs non-oxPhos DLBCL subtype. The good performance of the constructed predictive model was evaluated by prediction on unknown samples with sensitivity and specificity of 98%. CONCLUSIONS: We present a model of a classification of B cell lymphomas based on label-free Raman spectral imagines, capable of automatic and efficient identification of DLBCL cells spectra. In addition, we describe each lymphoma subtype by its unique spectral profile, linking it to biochemical features. Acknowledgements. The studies were performed as a part of the “ Label-free and rapid optical imaging, detection and sorting of leukemia cells” project carried out within the Team-Net programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

A Phase I Study of Copanlisib and Venetoclax in Patients with Relapsed or Refractory Diffuse Large B-Cell Lymphoma

Introduction: Diffuse large B-cell lymphoma (DLBCL) is a genomically heterogenous disease, which can be classified into distinct subgroups or clusters (Chapuy et al., Nat. Medicine, 2018, Wright et al., Cancer Cell, 2020). Leveraging knowledge of genetic alterations to design novel personalized treatment strategies may improve outcomes. Preclinical data suggest that the pan-phosphoinositide 3-kinase (PI3K) inhibitor, copanlisib, plus the BCL-2 inhibitor, venetoclax, are synergistic in genetically defined, high-risk, subtypes of DLBCL (Bojarczuk et al., Blood, 2019). We therefore hypothesized that copanlisib plus venetoclax would be an active treatment for patients with relapsed/refractory DLBCL, particularly in those with perturbed B-cell receptor (BCR)/PI3K signaling, which were predicted to be enriched in the relapsed setting. Here, we report the results of a phase I study of copanlisib plus venetoclax for patients (pts) with R/R DLBCL. Methods: This is an investigator-initiated, phase I, multicenter trial (NCT04572763) with the primary objective of determining the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of copanlisib and venetoclax in pts with R/R DLBCL. A standard 3+3 dose escalation design was employed, in which pts were treated with 60 mg IV copanlisib on days 1, 8, and 15 in addition to escalating doses of daily venetoclax for each 28-day cycle. During cycle 1, a venetoclax dose ramp-up was performed in the outpatient setting to a target dose of 200 mg (dose level (DL) +1), 400 mg (DL +2) and 800 mg (DL +3). The DLT observation period was the first cycle. Patients with progressive disease prior to the end of the DLT window were replaced. Key eligibility criteria included a confirmed diagnosis of DLBCL or high-grade B-cell lymphoma (HGBCL), relapse after or not a candidate for autologous stem cell transplant or chimeric antigen receptor (CAR) T-cell therapy, and adequate hematologic and organ function. Key exclusion criteria were poorly controlled hypertension or diabetes. CTCAE v5 and Lugano criteria were used to evaluate toxicity and efficacy, respectively. Results: As of July 21, 2023, 12 pts were evaluable. Patients were treated in DL +1 (n=5), DL +2 (n=3), and DL +3 (n=4). The median age was 62 years (range 24-87). Eighty three percent were male. Median number of prior treatments was 6 (range 1-11) and 10 pts had received prior CAR T-cell therapy. No DLTs were observed, though 2 pts in DL +3 were not able to escalate to 800 mg due to recurrent neutropenia and noncompliance, respectively. The most common toxicities were neutropenia (50%, all gr3/4), thrombocytopenia (42%, 8% ≥ gr3), nausea (33%, 8% ≥ gr3), fatigue (25%, 8% ≥ gr3), and headache (25%, 8% ≥ gr3) (Figure). Hyperglycemia occurred in 2 pts (both gr1). Serious adverse events occurred in 50% of pts and included fracture (n=1), hypoxia (n=2), and death (n=3), which were not related to study treatment. The deaths were related to progressive disease. Fifty percent of pts required drug holds (n=5 for venetoclax, n=5 for copanlisib). Sixty percent required growth factor support. There were no cases of laboratory or clinical tumor lysis syndrome. Sponsor support was withdrawn prior to completion of enrollment to DL+3, but DL+2 (copanlisib plus 400 mg venetoclax) was safe and well tolerated. The overall response rate (ORR) and complete metabolic response (CMR) rate was 8% (90% CI: 0 - 34%), as 1 pt in DL +1, who had received &amp;gt;5 lines of prior therapy, achieved a CMR, with response lasting 3 months. The median follow-up was 9.1 months. The median overall survival (OS) and progression-free survival (PFS) were 3.5 and 1.8 months, respectively. Conclusions: Copanlisib plus venetoclax is feasible and safe in pts with R/R DLBCL, with no DLTs observed. While it is possible that copanlisib plus venetoclax is active in select genomic subgroups of DLBCL, there was limited activity in this unselected, heavily pre-treated patient population.

Clinical Impact of the Spatial Organization of the Immune Tumor Microenvironment in Diffuse Large B-Cell Lymphoma

Introduction: Recent analyses of diffuse large B-cell lymphoma (DLBCL) have highlighted the clinical importance of immune tumor microenvironment (iTME), and based on the composition of the iTME, different DLBCL subtypes have been proposed (Kotlov et al. Cancer Discov. 2021, Steen et al. Cancer Cell. 2021). However, studies have mainly focused on the impact of different cell type proportions, whereas the clinical importance of their spatial organization has remained unclear. Materials and Methods: We used 12-plex immunohistochemistry panel to characterize B cells (CD20), T cells (CD3, CD4, CD8, FOXP3), macrophages (CD68, CD163), and immune checkpoint molecules (PD-1, PD-L1, CD96) from FFPE samples of 107 DLBCL patients treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP)- like immunochemotherapy. We performed image processing using the Ilastik and CellProfiler softwares, and segmented nuclei with a pretrained deep learning segmentation model. Using histoCAT software, we quantified marker intensities for each cell, phenotyped the cells with the Phenograph algorithm, and, finally, performed a neighborhood analysis to recognize the cell types that neighbor or avoid each other. We correlated the findings with patient demographics and survival. Results: In total,we analyzed 739 825 single cells (median 7127 per sample; range 1640 - 12705) and discovered 16 different metaclusters, which included various T helper cell, cytotoxic T cell, regulatory T cell (Treg), M1 and M2 like macrophage, and B cell subgroups. Samples varied greatly in their immune cell composition, with a median proportion of B cells, T cells and macrophages being 48.0 %, 19.9 %, and 10.2 %, respectively. We divided the samples according to their iTME constitution using K means clustering. As expected, samples were split into non-inflamed (37 %) and inflamed iTME subgroups, the latter dominated by T cells (22 %) and M2 macrophages (40 %). However, there was no significant difference in survival between the subgroups. Neighborhood analysis revealed several interaction patterns, such as lymphoma cells favoring neighboring with other lymphoma/B cells. Interestingly, when T cells and, especially cytotoxic T cells, in the inflamed iTME neighbored with PD-L1/PD-1 negative B cells, the outcome was favorable (OS; p &amp;lt; 0.05; Figure 1A), independent of the IPI and cell-of-origin. In contrast, when B cells expressed PD-L1 or PD-1, there was no association with survival. Within the iTME, CD4+ T helper cells often neighbored with other CD4+ T cells, including Tregs, as well as cytotoxic T cells, and to a lesser extent macrophages. Cytotoxic T cells commonly neighbored with other cytotoxic T cells, but also with macrophages and T helper cells, whereas M1 and M2 like macrophages frequently neighbored with other M1 and M2 like macrophages, respectively, as well as T cells. When we clustered the cases according to how often M2 macrophages neighbored other immune cells, we identified a group of cases, where M2 macrophages accumulated around T cells. Notably, this iTME pattern translated to unfavorable outcome (OS; p &amp;lt; 0.05; Figure 1B). Conclusions: Our data reveal clinically significant interaction patterns between B and T cells, as well as between macrophages and T cells in the iTME of DLBCL.

Phase II Clinical Trial of R-CHOP Combined with Lenalidomide in the First-Line Treatment for Patients with High Risk Diffuse Large B Cell Lymphoma

Introduction: Diffuse large B cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma (NHL). Currently, R-CHOP (Rituximab-Cyclophosphamide, Epirubicin, Vincristine and Prednisone) is world-widely used in the first-line treatment for DLBCL, producing a long-term survival rate of approximately 60%. The unmet need is focused on high-risk DLBCL patients, having high International Prognostic Index (IPI) scores, non-germinal center-like B cell (non-GCB) origin, double expression of MYC and BCL2 protein, or TP53 aberrations. Lenalidomide is an active agent in the activated B cell-like (ABC) subtype of DLBCL by increasing interferon-stimulated gene transcription and activation of immunomodulatory mechanisms. At present, it has been approved for the treatment of multiple myeloma with good efficacy and safety. This is a prospective single arm, multi-center, phase II clinical trial and the goal of our trial is to assess the efficacy and safety of R-CHOP combined with lenalidomide in the first-line treatment for patients with high risk diffuse large B cell lymphoma. Methods: We did an investigator-initiated, open-label, phase 2 trial at the Affiliated Cancer Hospital of Zhengzhou University in China. Patients were eligible if they were aged over 18 years, had histologically confirmed DLBCL. The key inclusion criteria included untreated and medium to high /high risk DLBCL (International Prognostic Index (IPI) score 3-5, aaIPI score 2-3 or NCCN-IPI score≥ 4/ Immunohistochemical staining of double expression (BCL2 ≥ 50% and C-MYC ≥ 40%) or P53 protein mutation positive ≥ 50%). Participants in RL-CHOP cohort were given Lenalidomide orally 25 mg per day on days 1 through 10 of each cycle and delivered concomitantly with standard dose R-CHOP-21 chemotherapy. We also set another group treated with conventional R-CHOP-21 used as a matched contemporary cohort. The primary clinical endpoint was 2-year progression-free survival (PFS). The secondary efficacy endpoints were objective response rate (ORR), 2-year overall survival (OS) and safety. This trial is registered with ClinicalTrials.gov, number NCT04214626. Results: Between December 2019 and January 2023, 63 patients with untreated DLBCL enrolled and were evaluable in the investigator-initiated phase II study. The median age was 59 years (range: 25-76 years); 49.2% of patients were older than age 60 years. 46 of 63 patients (73.02%) had a ECOG score of 0-1; 43 of 63 patients (68.25%) had stage III-IV disease, and 33 of 63 patients (52.38%) had ≥2 extranodal localizations. The IPI score was high-intermediate and high in 73.02% (46/63) of patients. 51 of 63 patients (80.95%) were non-GCB subtype; 25 of 63 patients (39.68%) were double expressor. The TP53 protein was positive in 34.55% (19/55) of patients. In RL-CHOP cohort, the complete response rate was 80.95%, with overall response rate achieving 100%. The median follow-up time was 18.25 (3.73-42) months, median PFS and OS were not reached, and 2-year PFS and OS was 71.1% (+-6.7) and 87.4% (+-4.5), respectively. Comparing with historical matched contemporary cohort, the 2-year PFS and OS rates were improved for medium to high /high risk DLBCL. The most common adverse events of treatment were hematologic toxicities. Grade 3/4 AEs occurred in ≥20% patients were neutropenia, leukopenia, anemia, thrombocytopenia and decreased serum potassium, without bleeding complications. No grade 4 non-hematological adverse event was reported. Conclusion: RL-CHOP is effective and safe in patients with newly diagnosed DLBCL with high risk. Relevance of molecular biomarkers on RL-CHOP response warrants further investigation in DLBCL.

Molecular Determinants of Response to Mosunetuzumab Plus CHOP in Patients with Previously Untreated (1L) Diffuse Large B Cell Lymphoma (DLBCL)

Co-lead authors: Habib Hamidi and Patrick Kimes Background: Standard induction therapy cures ~60% of patients (pts) with DLBCL; however, most remaining non-responders die from lymphoma, highlighting a need for improved first-line treatments. Biomarkers that can reliably assess the efficacy of new treatments are lacking in clinical practice. Mosunetuzumab (Mosun) is a CD20xCD3 T cell engaging bispecific antibody that redirects T cells to eliminate malignant B cells. In a Phase Ib/II trial (NCT03677141), Mosun combined with CHOP chemotherapy (M-CHOP) induced high response rates in pts with 1L DLBCL (ORR 87.5%, CR 85%; Phillips et al. ASH 2020). Here, we sought to identify biomarkers associated with M-CHOP response. Methods: Pts with 1L DLBCL receiving M-CHOP (six cycles) in NCT03677141 and with available biomarker data were included. Bulk RNASeq data from pre-treatment tumor biopsies were analyzed using gene set variation for tumor intrinsic pathways and xCell cell-type enrichment for immune and stromal cell types. High or low expression of each gene signature was determined by median cut-off. Cox regression examined associations between gene signatures and PFS. To assess specificity to M-CHOP, gene signatures were examined in pre-treatment biopsies from pts treated with rituximab + CHOP chemotherapy (R-CHOP) in GOYA (NCT01287741; Sehn et al. J Hematol Oncol 2020). Peripheral blood biomarkers were evaluated by whole blood flow cytometry and plasma cytokines by ELISA. Circulating tumor (ct) DNA levels were measured at baseline and on treatment using the AVENIO NHL CAPP-Seq assay (Stokowski et al. ASH 2022). Results: In M-CHOP-treated pts (n=33), gene expression analysis revealed two types of signatures associated with PFS: tumor immune microenvironment signature (TIMS) and tumor intrinsic proliferation signature (TIPS; Figure). High TIMS expression (e.g., CD4+ T cells) was associated with significantly longer PFS than low expression. Conversely, pts with high TIPS expression (e.g., MYC targets, DNA repair) had shorter PFS than those with low expression. These biological associations were specific to M-CHOP and were not observed in R-CHOP-treated pts. Consistent with the tumor-gene expression analysis, immune profiling of peripheral blood suggested that in pts treated with M-CHOP, a higher T cell baseline count was associated with a trend for prolonged PFS compared with pts with a lower baseline count. A higher count of circulating suppressive T cells was also associated with shortened PFS. Early on-treatment pharmacodynamic (PD) changes were observed following the first Mosun dose, including: induction of TNFα and IL-6, transient margination of CD4+ and CD8+ T cells, and activation of CD4+ and CD8+ T cells. The magnitude of the PD effects were similar to observations with Mosun monotherapy in pts with relapsed or refractory DLBCL (Hernandez et al. ASH 2019), confirming that Mosun maintains its immune activation effect when combined with CHOP in pts with 1L DLBCL. High baseline ctDNA levels were associated with high IPI score (n=32, Wilcoxon rank-sum p=0.007), ABC/non-GCB DLBCL subtype (n=30, p=0.033), and high lactate dehydrogenase (n=32, p=0.004). Pts with baseline ctDNA levels &amp;lt; median had a modest trend for improved PFS compared with pts &amp;gt; median (unadjusted hazard ratio [HR]=0.57 [95% CI 0.16, 1.75]; IPI-adjusted HR=0.72 [95% CI 0.16, 3.20]). The proportion of pts with undetectable ctDNA increased with each M-CHOP cycle (C) (C2 day [D]1, 25% [8/32]; C3D1, 69% [20/29]; C5D1, 88% [22/25]). Pts with undetectable ctDNA at any on-treatment time point (C2D1, C3D1, C5D1) had superior PFS compared with pts with detectable ctDNA (unadjusted HR=0.12 [95% CI 0.03, 0.52]; IPI adjusted HR=0.14 [95% CI 0.03, 0.61]). Conclusions: Consistent with the Mosun mode of action, pts with high expression of TIMS and those with high levels of circulating T cells had improved PFS versus pts with low expression. In contrast, there was no association between immune cell signatures and PFS in pts treated with R-CHOP, suggesting that the different mode of action of rituximab versus Mosun may influence this. Future studies are warranted to confirm the potential clinical utility of immune cell signatures as biomarkers associated with CD20xCD3 bispecifics' activity.

The Immune Checkpoint Siglec-15 in Promoting Immune Dysregulation in Non-Hodgkin's Lymphomas

Outcomes for patients with relapsed or refractory (r/r) hematologic malignancies remain dismal despite therapeutic advances over the last several years. This is particularly striking in patients with r/r aggressive mature B-cell lymphomas such as Diffuse Large B-cell Lymphoma (DLBCL) and Burkitt's Lymphoma (BL). With use of intensive multiagent chemo-immunotherapy, DLBCL, which accounts for approximately 10-20% of pediatric Non-Hodgkin's Lymphoma (NHL) cases and is the most common subtype in adults, is highly curable with 5-year event free survival &amp;gt;75%. However, approximately 30-40% of adults diagnosed with DLBCL will become refractory to or relapse after first-line treatment. Yet even with intensive salvage regimens, r/r DLBCL remains a clinical challenge. There remains an unmet need to identify specific defects in the immune response, among other pathways, that drive lymphomagenesis that will in turn inform the development of novel therapeutics to improve survival in this population of patients. Siglec-15 (Sig-15), a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, recent reports have demonstrated inhibition of Sig-15, has a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies and acute leukemias, a role for Siglec-15 in promoting disease progression in aggressive B-cell lymphomas has not yet been described. We have found higher expression of SIGLEC15 in DLBCL cell lines compared to normal B cells at the RNA level, through analyses of the publicly available ONCOMINE database. This was confirmed at the protein level by western blot demonstrating higher Sig-15 expression in various lymphoma cell lines (RAJI, SUDHL6, OCI-LY10, HBL-1) compared to healthy donor PBMCs. Further, immunohistochemistry evaluating Sig-15 expression was performed on a tumor microarray consisting of 139 cases of DLBCL from adult patients along with validation samples from 15 primary pediatric lymphoma cases (DLBCL, BL and Anaplastic Large Cell Lymphoma (ALCL)). Sig-15 was found to be highly expressed, with histologic scores ranging from weak (1+) to strong (3+) in 124 out of the 139 adult cases (Figure 1A) and in all pediatric lymphoma samples but with distinct staining patterns observed in the aggressive B-cell lymphomas. Specifically, Sig-15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt's lymphoma. Expression is more variable in ALCL, primarily elevated in the cytoplasm at low levels and/or in macrophages (Figure 1A). There is heterogeneity in Sig-15 expression with preliminary analysis of RNA-seq data from a cohort of 693 DLBCL patient samples demonstrates higher levels in the germinal center B-cell (GCB) versus activated B-cell molecular (ABC) subtype of DLBCL when compared to healthy controls (Kruskal-Wallis, *p = 0.041). Finally, using a well-established murine lymphoma model, A20 cells were stably transduced with control, non-silencing shRNA (shNS) or shRNA against Sig-15 and injected into un-irradiated immune competent (wild type, WT) or immune deficient ( Rag1 -/-) BALB/C mice and monitored for signs of lymphoma development. Knockdown of Sig-15 in A20 cells abrogates disease progression in WT but not immune deficient mice (Log-rank, **p &amp;lt;0.0001, n=10 mice/group), consistent with a role for Sig-15 in immune evasion in lymphoma (Figure 1B). Together, these data implicate Sig-15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells.

Development of Cytokine Release Syndrome Is Associated with Superior Progression-Free Survival in DLBCL Patients Receiving Axi-Cel or Liso-Cel

Introduction: Chimeric antigen receptor T-cell therapy (CAR-T) targeting CD19 is a new standard of care for diffuse large B-cell lymphoma (DLBCL) patients with primary refractory disease, early relapse after chemoimmunotherapy, or multiply relapsed disease. Three CD19-directed CAR-T products, axicabtagene ciloleucel (axi-cel), tisagenlecleucel (tisa-cel), and lisocabtagene maraleucel (liso-cel), are FDA approved for relapsed/refractory (R/R) DLBCL and may cure approximately 30-40% of patients. Cytokine release syndrome (CRS), a systemic inflammatory response associated with CAR-T expansion, is a common early toxicity occurring in 42-93% of DLBCL patients in the pivotal phase 2 trials. We hypothesized that development of CRS may be associated with post-CAR-T efficacy outcomes. Methods: We retrospectively evaluated post-CAR-T outcomes for 57 consecutive patients with R/R DLBCL who received axi-cel or liso-cel infusion at the University of California San Francisco between June 2018 and December 2022. Early post-CAR-T toxicities including CRS and immune effector cell-associated neurotoxicity syndrome (ICANS) were recorded using ASTCT consensus grading. Post-CAR-T efficacy endpoints including overall response rate (ORR), complete remission (CR) rate (best response), progression-free survival (PFS), and overall survival (OS) were evaluated for the entire cohort and compared between patients who developed any grade CRS or no CRS after CAR-T infusion. Of note, patients with high-grade B-cell lymphoma or who received tisa-cel were excluded from this study to eliminate potential confounding given poorer outcomes in these subgroups and imbalance in their relative proportions between the CRS and no CRS cohorts. Results: For the entire cohort, the median age was 63 (range 26-81) and 59% of patients were male. Diagnoses included de novo DLBCL (65%), transformed follicular lymphoma (30%), and primary mediastinal B-cell lymphoma (5%). Patients had a median of 2.5 systemic therapies (range 1-8) prior to CAR-T infusion. CAR-T products included axi-cel (70%) or liso-cel (30%). 82% of patients received fludarabine and cyclophosphamide lymphodepletion, and the remaining 18% received bendamustine (during the national fludarabine shortage). Forty-one patients (72%) experienced CRS (grade 1: 35%, grade 2: 37%), and 16 patients (28%) never developed CRS. Seventeen patients (30%) developed ICANS (grade 2: 12%, grade 3: 16%, grade 4: 2%), which occurred exclusively among patients in the CRS cohort. 93% of patients in the CRS cohort received at least one dose of tocilizumab. Baseline characteristics were well balanced between the CRS and no CRS groups with no significant differences in age, sex, performance status, DLBCL subtype, number of prior therapies, CAR-T product administered, lymphodepletion administered, or lactate dehydrogenase at the time of infusion. For the entire cohort, the ORR and CR rate were 86% and 70%, respectively. With a median follow-up of 1.1 years (range 0.2-4.5 years), the 1-year PFS and OS estimates were 61% and 91%, respectively. Median PFS was 1.7 years and median OS was not reached. Outcomes were similar for patients receiving axi-cel and liso-cel (1-year PFS 62% vs 60%, p=0.94). For patients in the CRS cohort vs no CRS cohort, the ORR was 90% vs 75% (p=0.14) and CR rate was 73% vs 63%, respectively (p=0.44). Patients in the CRS cohort had superior PFS compared to patients with no CRS (1-year PFS 70% vs 38%, HR 0.36, 95% CI 0.14-0.98, logrank p=0.011), but there was no significant difference in OS ( Figure 1). There was no significant difference in PFS between patients with grade 1 vs grade 2 CRS (1-year PFS 64% vs 75%, p=0.46). Conclusions: Although limited in sample size, our study suggests that development of CRS may be associated with more favorable post-CAR-T efficacy including superior PFS compared to patients with no CRS after axi-cel or liso-cel infusion. The more favorable outcomes in patients experiencing CRS may reflect better CAR-T proliferation and cytotoxicity compared to patients with no CRS. These findings suggest a relationship between early post-CAR-T toxicity and efficacy outcomes but require validation in larger datasets.

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.

A CD5 Gene Signature Identifies Diffuse Large B-Cell Lymphomas Sensitive to Brutonʼs Tyrosine Kinase Inhibition

Introduction: Diffuse large B-cell lymphomas (DLBCLs) with a non-germinal center B cell-like (non-GCB) cell-of-origin are frequently driven by genetic alterations that culminate in constitutive B-cell receptor (BCR) signaling, which has inspired the exploration of Bruton's tyrosine kinase inhibitors (BTKi) in these lymphomas. However, the phase III PHOENIX study that randomized untreated, non-GCB DLBCL patients to R-CHOP plus placebo or ibrutinib failed to meet its primary endpoint of event-free survival (Younes et al. 2019), which suggests that cell-of-origin alone is an insufficient biomarker to predict BTKi sensitivity in DLBCL. More recently, a DLBCL genetic classifier termed LymphGen has identified distinct subtypes (MCD and N1) of non-GCB DLBCL that benefit from the addition of BTKi to R-CHOP (Wilson et al. 2021). However, genetic classifiers are complex and difficult to implement in routine clinical settings and may fail to capture all DLBCLs that benefit from BTKi. Therefore, we sought to identify a straightforward biomarker of BTKi responsiveness in DLBCL with greater precision than cell-of-origin but with broader inclusivity than current genomic platforms, such as LymphGen. We hypothesized that CD5 - a surrogate marker of BCR activation - may effectively identify BCR-driven, non-GCB DLBCLs that are sensitive to BTKi therapy, and evaluated the extent to which CD5 protein expression and a transcriptionally defined CD5 gene signature could accurately identify BCR-activated DLBCLs with potential susceptibility to BTKi-based therapies. Methods: CD5 immunohistochemistry (IHC) was performed on a cohort of 406 diagnostic DLBCL samples, which were considered CD5+ if &amp;gt;=30% of lymphoma cells exhibited unequivocal membranous staining. A majority of DLBCL samples had available RNA-sequencing and targeted mutational sequencing data. A comparison of differentially expressed genes between CD5+ and CD5- DLBCLs was performed in order to construct a 60-gene CD5 signature (CD5sig), which was applied to large genomic DLBCL datasets, including pre-treatment biopsies from patients enrolled on PHOENIX (n = 584) to evaluate the utility of the CD5sig in identifying DLBCLs that benefitted from the addition of ibrutinib to R-CHOP. Results: Twenty-six of 406 DLBCLs were identified as CD5+ by IHC (6% of all DLBCLs; 12% of non-GCB DLBCLs). CD5 IHC+ DLBCLs were majority non-GCB cell-of-origin and were associated with inferior progression-free survival (PFS) to R-CHOP (50% 3-year PFS), compared with CD5 IHC- DLBCLs, consistent with previous reports. Gene set enrichment analysis revealed that CD5 IHC+ DLBCLs exhibited transcriptional features of BCR activation, and mutational analysis demonstrated that CD5 IHC+ DLBCLs were enriched for CD79B BCR-activating mutations known to correlate with BTKi sensitivity. Many CD5 IHC+ DLBCLs, however, lacked canonical BCR-activating mutations or were classified as “Other” by LymphGen. A CD5 gene signature (CD5sig; Figure 1A) was developed that recapitulated these findings in independent DLBCL datasets (NCI, Duke), where ~13% of non-GCB DLBCLs were classified as CD5sig+. Together, these results suggest that CD5 signature expression captures DLBCLs with both a genetic and non-genetic basis for BCR dependence. Supporting this notion, CD5sig+ DLBCL patients (&amp;lt; 60 years) derived a selective and striking event-free and overall survival advantage from the addition of ibrutinib to R-CHOP in the PHOENIX study ( Figure 1B), independent of LymphGen classification. Conclusions: We demonstrate that CD5 IHC and a novel CD5 gene signature identify high-risk, BCR-driven DLBCLs. Importantly, the CD5 signature also identifies DLBCL patients with a selective survival advantage to BTK inhibitor-based therapy, independent of LymphGen classification. In conclusion, the CD5 signature expands upon LymphGen classification as a biomarker of BTKi response by accurately identifying DLBCLs with both genetic and non-genetic bases for BTKi response. The CD5 signature and/or CD5 IHC should be prospectively evaluated in BTKi-based clinical trials for non-GCB DLBCLs.

Oligomannose-Type Glycans in the Antigen Binding Site Identify Origin and Prognosis of Diffuse Large B Cell Lymphoma

The acquisition of N-glycosylation sites (AGS) in the complementarity-determining region (CDR) of the tumor immunoglobulin (Ig) is an early clonal requirement of classic follicular lymphoma (FL). In FL, the AGS are occupied by oligomannose-type glycans, which mediate lymphoma Ig interaction with the microenvironmental lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN). This interaction induces a persistent low-level signal, favouring adhesion and ultimately survival and growth of the lymphoma cells in their protected environment . Oligomannose-type glycans can also occupy the CDR-located AGS of a subset of germinal-centre B cell-like (GCB) diffuse large B-cell lymphomas (DLBCL) but the consequences of AGS in DLBCL are less known. We determined the frequency, distribution, and composition of the glycan structures occupying the AGS in DLBCL using a discovery-validation approach with two large independent international public cohorts (BCC, dataset ID: EGAD00001003783; NCI accession phs001444.v1.p1), following our RNAseq Ig assembly pipeline (Ig-Seq-R) and high-resolution mass-spectrometry. Glycan types were compared to our FL cohort (12 samples) and correlated with DLBCL cell of origin (COO), genetic, clinical characteristics and outcome. The full IGHV-IGHD-IGHJ-IGHC transcript sequences were obtained from 251 DLBCL of the BCC, and 339 of the NCI cohort. Sample distribution by COO, clinical or molecular features, LymphGen subtype, progression-free survival (PFS) and overall survival (OS) were not different from the extended public cohorts. AGS were observed in 48-55% GCB-DLBCL, with 84-85% of them located in the CDR. Overall, frequency of CDR-located AGS contrasted dramatically between GCB-DLBCL (41-46%) and activated B-cell like (ABC)-DLBCL (7-10%). Also, within GCB-DLBCL, CDR-located AGS associated mostly with the EZB subtype (68-73%). However, only 66-61% EZB were AGS+ve, all of which were CDR+ve, while the remaining 34-39% had no AGS (AGS-ve EZB). The glycan structures of the synthetic F(ab)s from 35 AGS+ve DLBCL and 12 FL were analyzed by site-specific mass spectrometry. The DLBCL comprised 12 AGS+ve EZB and 23 AGS+ve non-EZB (14 CDR+ve, 9 located in the framework region). Glycan analysis revealed that all AGS+ve EZB and FL were invariably occupied by oligomannose-type glycans (Ig-Mann+ve DLBCL). In contrast, the other AGS+ve DLBCL were not occupied by oligomannose type glycans. They were either occupied by complex glycans (the remaining non-EZB GCB-DLBCL) or not occupied (preferentially the ABC-DLBCL), irrespective of AGS location. These data indicated that CDR+ve EZB Ig were universally and exclusively Ig-Mann+ve DLBCL, share the COO of FL, and have likely been influenced by an environmental driver distinct from the other GCB-DLBCL and ABC-DLBCL. We compared the clinical behavior of Ig-Mann+ve DLBCL versus other DLBCL subtypes including AGS-ve EZB and the other non-EZB GCB-DLBCL and ABC-DLBCL. In both the BCC and NCI cohorts, Ig-Mann+ve DLBCL status identified the subset with the worst PFS and OS of all GCB-DLBCL, not different from ABC-DLBCL (Figure 1A). This contrasted with the other GCB-DLBCL (AGS-ve/EZB and AGS+ve/non-EZB), which all shared an extremely good PFS and OS (Figure 1A). Univariate and multivariate analyses revealed that Ig-Mann+ve status was an independent prognostic factor for PFS and OS (Figure 1B). Gene expression profile analysis revealed that Ig-Mann+ve status associated with an enrichment of MYC, PI3K-Akt-mTORC1, pro-survival, and cell cycle pathways, while proinflammatory and apoptotic pathways were decreased in the Ig-Mann+ve DLBCL compared to AGS-ve/EZB and AGS+ve/non-EZB, independently of MYC translocations and double-hit signature. These data indicate identical COO of a EZB subset with FL cells, and point to a highly selective chronic environmental pressure on Ig-Mann+ve DLBCL negatively affecting patients survival. The combination of Ig gene analysis with the lymphGen classification can predict Ig glycan structure and provide a new fundamental approach to identifying the most aggressive GCB-DLBCL subtype, and the precise environmental tumour interaction (DC-SIGN:Ig-Mann) to intercept therapeutically early in the natural history of Ig-Mann+ve DLBCL.