Selective deletion of E3 ubiquitin ligase FBW7 in VE-cadherin-positive cells instigates diffuse large B-cell lymphoma in mice in vivo.

During the maturation of hematopoietic stem/progenitor cells (HSPCs) to fully differentiated mature B lymphocytes, developing lymphocytes may undergo malignant transformation and produce B-cell lymphomas. Emerging evidence shows that through the endothelial-hematopoietic transition, specialized endothelial cells called the hemogenic endothelium can differentiate into HSPCs. However, the contribution of genetic defects in hemogenic endothelial cells to B-cell lymphomagenesis has not yet been investigated. Here, we report that mice with endothelial cell-specific deletion of Fbw7 spontaneously developed diffuse large B-cell lymphoma (DLBCL) following Bcl6 accumulation. Using lineage tracing, we showed that B-cell lymphomas in Fbw7 knockout mice were hemogenic endothelium-derived. Mechanistically, we found that FBW7 directly interacted with Bcl6 and promoted its proteasomal degradation. FBW7 expression levels are inversely correlated with BCL6 expression. Additionally, pharmacological disruption of Bcl6 abolished Fbw7 deletion-induced B-cell lymphomagenesis. We conclude that selective deletion of E3 ubiquitin ligase FBW7 in VE-cadherin positive endothelial cells instigates diffuse large B-cell lymphoma via upregulation of BCL6 stability. In addition, the mice with endothelial cell-specific deletion of Fbw7 provide a valuable preclinical platform for in vivo development and evaluation of novel therapeutic interventions for the treatment of DLBCL.

Ferroptosis mechanisms and its novel potential therapeutic targets for DLBCL.

Diffuse large B-cell lymphoma (DLBCL), a heterogeneous lymphoid malignancy, poses a significant threat to human health. The standard therapeutic regimen for patients with DLBCL is rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP), with a typical cure rate of 50-70%. However, some patients either relapse after complete remission (CR) or exhibit resistance to R-CHOP treatment. Therefore, novel therapeutic approaches are imperative for managing high-risk or refractory DLBCL. Ferroptosis is driven by iron-dependent phospholipid peroxidation, a process that relies on the transition metal iron, reactive oxygen species (ROS), and phospholipids containing polyunsaturated fatty acids-containing phospholipids (PUFA-PLs). Research indicates that ferroptosis is implicated in various carcinogenic and anticancer pathways. Several hematological disorders exhibit heightened sensitivity to cell death induced by ferroptosis. DLBCL cells, in particular, demonstrate an increased demand for iron and an upregulation in the expression of fatty acid synthase. Additionally, there exists a correlation between ferroptosis-associated genes and the prognosis of DLBCL. Therefore, ferroptosis may be a promising novel target for DLBCL therapy. In this review, we elucidate ferroptosis mechanisms, its role in DLBCL, and the potential therapeutic targets in DLBCL. This review offers novel insights into the application of ferroptosis in treatment strategies for DLBCL.

LncRNA CHROMR/miR-27b-3p/MET axis promotes the proliferation, invasion, and contributes to rituximab resistance in diffuse large B-cell lymphoma.

Long non-coding RNAs (LncRNAs) could regulate chemoresistance through sponging microRNAs (miRNAs) and sequestering RNA binding proteins. However, the mechanism of lncRNAs in rituximab resistance in diffuse large B-cell lymphoma (DLBCL) is largely unknown. Here, we investigated the functions and molecular mechanisms of lncRNA CHROMR in DLBCL tumorigenesis and chemoresistance. LncRNA CHROMR is highly expressed in DLBCL tissues and cells. We examined the oncogenic functions of lncRNA CHROMR in DLBCL by a panel of gain-or-loss-of-function assays and in vitro experiments. LncRNA CHROMR suppression promotes CD20 transcription in DLBCL cells and inhibits rituximab resistance. RNA immunoprecipitation, RNA pull-down, and dual luciferase reporter assay reveal that lncRNA CHROMR sponges with miR-27b-3p to regulate mesenchymal-epithelial transition factor (MET) levels and Akt signaling in DLBCL cells. Targeting the lncRNA CHROMR/miR-27b-3p/MET axis reduces DLBCL tumorigenesis. Altogether, these findings provide a new regulatory model, lncRNA CHROMR/miR-27b-3p/MET, which can serve as a potential therapeutic target for DLBCL.

Dihydrocelastrol induces cell death and suppresses angiogenesis through BCR/AP-1/junb signalling in diffuse large B cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Although treatment options have improved, a large proportion of patients show low survival rates, highlighting an urgent need for novel therapeutic strategies. The aim of this study was to investigate the efficacy of the new small-molecule compound dihydrocelastrol (DHCE), acquired through the structural modification of celastrol (CE), in the treatment of DLBCL. DHCE showed potent anti-lymphoma efficacy and synergistic effects with doxorubicin. DHCE triggered DLBCL cell apoptosis and G0/G1-phase blockade, thereby hindering angiogenesis. DHCE inhibited B-cell receptor cascade signalling and Jun B and p65 nuclear translocation, thereby suppressing pro-tumourigenic signalling. Finally, DHCE exerted lower toxicity than CE, which showed severe hepatic, renal, and reproductive toxicity in vivo. Our findings support further investigation of the clinical efficacy of DHCE against DLBCL.

Tumoural Pellino-1 expression and Pellino-1-expressive cytotoxic T-cells are associated with poor prognosis in diffuse large B-cell lymphoma.

Pellino-1 plays a role in regulating inflammation and immune responses, and its effects on tumours are complex, with different outcomes reported in various studies. Additionally, the role of Pellino-1 in diffuse large B-cell lymphoma (DLBCL) has not been thoroughly investigated. We aimed to examine the expression of Pellino-1 in tumour cells and tumour-infiltrating lymphocytes (TILs) separately and identify the clinicopathological significance of Pellino-1 expression in DLBCL. We evaluated Pellino-1 expression in 104 patients with DLBCL. The density of specific cell types was quantitatively analysed using digital image analysis after a multiplex immunofluorescence staining with Pellino-1, CD20, CD8, FOXP3, and PD-1. Pellino-1 expression was mostly observed in CD20+ tumour cells and CD8+ TILs. The high CD8+/Pellino-1+ group was significantly associated with the non-GCB subtype and higher numbers of Foxp3+ T-cells. Patients with high CD20+/Pellino-1+ and high CD8+/Pellino-1+ cell densities had significantly shorter event-free survival (EFS) rates. The multivariate Cox-regression analysis showed that CD20+/Pellino-1+ cell density and CD8+/Pellino-1+ cell density were independent poor prognostic factors for EFS. Furthermore, patients with low densities of both CD20+/Pellino-1+ and CD8+/Pellino-1+ cells demonstrated a prognosis superior to that of patients with high Pellino-1+ cell densities, either alone or in combination. Additionally, the multivariate analysis demonstrated that the combination of CD20+/Pellino-1+ and CD8+/Pellino-1+ cell densities was an independent prognostic factor for EFS and overall survival. Pellino-1 expression was observed in both tumour cells and TILs, particularly in cytotoxic T-cells, and was correlated with poor outcomes in DLBCL. Thus, Pellino-1 might have an oncogenic effect on DLBCL and might be a potential target for improving cytotoxic T-cell activity.

Targeting ZDHHC21/FASN axis for the treatment of diffuse large B-cell lymphoma.

S-palmitoylation is essential for cancer development via regulating protein stability, function and subcellular location, yet the roles S-palmitoylation plays in diffuse large B-cell lymphoma (DLBCL) progression remain enigmatic. In this study, we uncovered a novel function of the palmitoyltransferase ZDHHC21 as a tumor suppressor in DLBCL and identified ZDHHC21 as a key regulator of fatty acid synthetase (FASN) S-palmitoylation for the first time. Specifically, ZDHHC21 was downregulated in DLBCL, and its expression level was associated with the clinical prognosis of patients with DLBCL. In vitro and in vivo experiments suggested that ZDHHC21 suppressed DLBCL cell proliferation. Mechanistically, ZDHHC21 interacted with FASN and mediated its palmitoylation at Cys1317, resulting in a decrease in FASN protein stability and fatty acid synthesis, consequently leading to the inhibition of DLBCL cell growth. Of note, an FDA-approved small-molecule compound lanatoside C interacted with ZDHHC21, increased ZDHHC21 protein stability and decreased FASN expression, which contributed to the suppression of DLBCL growth in vitro and in vivo. Our results demonstrate that ZDHHC21 strongly represses DLBCL cell proliferation by mediating FASN palmitoylation, and suggest that targeting ZDHHC21/FASN axis is a potential therapeutic strategy against DLBCL.

HoLy-Net: Segmentation of histological images of diffuse large B-cell lymphoma.

Over the last years, there has been large progress in automated segmentation and classification methods in histological whole slide images (WSIs) stained with hematoxylin and eosin (H&E). Current state-of-the-art (SOTA) techniques are based on diverse datasets of H&E-stained WSIs of different types of predominantly solid cancer. However, there is a scarcity of methods and datasets enabling segmentation of tumors of the lymphatic system (lymphomas). Here, we propose a solution for segmentation of diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin's lymphoma. Our method applies to both H&E-stained slides and to a broad range of markers stained with immunohistochemistry (IHC). While IHC staining is an important tool in cancer diagnosis and treatment decisions, there are few automated segmentation and classification methods for IHC-stained WSIs. To address the challenges of nuclei segmentation in H&E- and IHC-stained DLBCL images, we propose HoLy-Net - a HoVer-Net-based deep learning model for lymphoma segmentation. We train two different models, one for segmenting H&E- and one for IHC-stained images and compare the test results with the SOTA methods as well as with the original version of HoVer-Net. Subsequently, we segment patient WSIs and perform single cell-level analysis of different cell types to identify patient-specific tumor characteristics such as high level of immune infiltration. Our method outperforms general-purpose segmentation methods for H&E staining in lymphoma WSIs (with an F1 score of 0.899) and is also a unique automated method for IHC slide segmentation (with an F1 score of 0.913). With our solution, we provide a new dataset we denote LyNSeC (lymphoma nuclear segmentation and classification) containing 73,931 annotated cell nuclei from H&E and 87,316 from IHC slides. Our method and dataset open up new avenues for quantitative, large-scale studies of morphology and microenvironment of lymphomas overlooked by the current automated segmentation methods.

Epstein-Barr virus-encoded EBNA2 downregulates ICOSL by inducing miR-24 in B-cell lymphoma.

ABSTRACT: Hematological malignancies such as Burkitt lymphoma (BL), Hodgkin lymphoma (HL), and diffuse large B-cell lymphoma (DLBCL) cause significant morbidity in humans. A substantial number of these lymphomas, particularly HL and DLBCLs have poorer prognosis because of their association with Epstein-Barr virus (EBV). Our earlier studies have shown that EBV-encoded nuclear antigen (EBNA2) upregulates programmed cell death ligand 1 in DLBCL and BLs by downregulating microRNA-34a. Here, we investigated whether EBNA2 affects the inducible costimulator (ICOS) ligand (ICOSL), a molecule required for efficient recognition of tumor cells by T cells through the engagement of ICOS on the latter. In virus-infected and EBNA2-transfected B-lymphoma cells, ICOSL expression was reduced. Our investigation of the molecular mechanisms revealed that this was due to an increase in microRNA-24 (miR-24) by EBNA2. By using ICOSL 3' untranslated region-luciferase reporter system, we validated that ICOSL is an authentic miR-24 target. Transfection of anti-miR-24 molecules in EBNA2-expressing lymphoma cells reconstituted ICOSL expression and increased tumor immunogenicity in mixed lymphocyte reactions. Because miR-24 is known to target c-MYC, an oncoprotein positively regulated by EBNA2, we analyzed its expression in anti-miR-24 transfected lymphoma cells. Indeed, the reduction of miR-24 in EBNA2-expressing DLBCL further elevated c-MYC and increased apoptosis. Consistent with the in vitro data, EBNA2-positive DLBCL biopsies expressed low ICOSL and high miR-24. We suggest that EBV evades host immune responses through EBNA2 by inducing miR-24 to reduce ICOSL expression, and for simultaneous rheostatic maintenance of proproliferative c-MYC levels. Overall, these data identify miR-24 as a potential therapeutically relevant target in EBV-associated lymphomas.

Optimization of Metabolic Tumor Volume as a Prognostic Marker in CAR T-Cell Therapy for Aggressive Large B-cell NHL.

BACKGROUND: CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has become a standard of care in relapsed/refractory (R/R) aggressive large B-cell non-Hodgkin lymphomas (B-NHL) though the majority of recipients do not receive durable disease benefit, prompting the need to better define risk factors for relapse/progression. OBJECTIVES: We performed a single-center, retrospective analysis of patients treated with commercial CAR T-cell therapy to evaluate the impact of tumor burden, as measured by whole-body metabolic tumor volume (MTV) from 18F fluorodeoxyglucose PET imaging, on treatment outcomes. STUDY DESIGN: Sixty-one patients treated with CAR T-cell therapy for R/R B-NHL between May 2016 and November 2021 were included. RESULTS: Using a receiver operating characteristic curve-based MTV optimization cutoff of 450 mL, 1-year progression-free survival (PFS) was 22% for high MTV versus 54% for low MTV (P < .01), and 1-year overall survival (OS) was 37% and 73%, respectively (P = .01). In a subset of 46 patients, residual MTV of less than 106 mL at the day 30 (D30) disease assessment was associated with significantly improved outcomes (1-year OS 85% vs. 13%, P < .01). Incorporation of pretreatment MTV to the International Prognostic Index (IPI) scoring system significantly distinguished 2-year PFS and OS outcomes by 3 risk groups. CONCLUSIONS: Our findings suggest that both pretreatment and D30 MTV are predictive of outcomes among R/R B-NHL patients treated with CAR T-cell therapy. These data indicate that efforts to reduce pretreatment tumor burden may improve longitudinal clinical outcomes. Furthermore, D30 postinfusion MTV quantification may aid clinicians in optimally identifying patients at high-risk for progression, and in whom closer disease monitoring should be considered. MTV also adds prognostic value to patients with high-risk IPI and holds promise for incorporation in novel risk scoring systems which can identify patients prior to CAR T-cell therapy at highest risk of adverse outcomes.

Tumor Location Relative to the Spleen Is a Prognostic Factor in Lymphoma Patients: A Demonstration from the REMARC Trial.

Baseline [18F]FDG PET/CT radiomic features can improve the survival prediction in patients with diffuse large B-cell lymphoma (DLBCL). The purpose of this study was to investigate whether characterizing tumor locations relative to the spleen location in baseline [18F]FDG PET/CT images predicts survival in patients with DLBCL and improves the predictive value of total metabolic tumor volume (TMTV) and age-adjusted international prognostic index (IPI). Methods: This retrospective study included 301 DLBCL patients from the REMARC (NCT01122472) cohort. Physicians delineated the tumor regions, whereas the spleen was automatically segmented using an open-access artificial intelligence algorithm. We systematically measured the distance between the centroid of the spleen and all other lesions, defining the SD of these distances as the lesion spread (SpreadSpleen). We calculated the maximum distance between the spleen and another lesion (Dspleen) for each patient and normalized it with the body surface area, resulting in standardized Dspleen (sDspleen). The predictive value of each PET/CT feature for progression-free survival (PFS) and overall survival (OS) was evaluated through univariate and multivariate time-dependent Cox models and Kaplan-Meier analysis. Results: In total, 282 patients (mean age, 68.33 ± 5.41 y; 164 men) were evaluated. The artificial intelligence algorithm successfully segmented the spleen in 96% of the patients. SpreadSpleen, Dspleen, and sDspleen were correlated neither with TMTV (Pearson ρ < 0.23) nor with IPI (Pearson ρ < 0.15). When median values were used as the cutoff, SpreadSpleen, Dspleen, and sDspleen all significantly classified patients into 2 risk groups for PFS and OS (P < 0.001). They complemented TMTV and IPI to classify the patients into 3 risk groups for PFS and OS (P < 0.001). Integrating SpreadSpleen, Dspleen, or sDspleen into a Cox model on the basis of TMTV, IPI, and TMTV combined with IPI significantly improved the concordance index for PFS and OS (P < 0.05). Conclusion: Baseline PET/CT features that characterize tumor spread and dissemination relative to the spleen strongly predicted survival in patients with DLBCL. Integrating these features with TMTV and IPI further improved survival prediction.

[18F-FDG PET/CT Metabolic Parameters and Circulating Tumour DNA Mutation Abundance in Diffuse Large B-Cell Lymphoma: Correlation and Survival Analysis].

OBJECTIVE: To investigate the correlation between 18Fluoro-deoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) metabolic parameters and peripheral blood circulating tumour DNA (ctDNA) in patients with diffuse large B-cell lymphoma (DLBCL), and the prognostic value of these two types of parameters in predicting progression-free survival (PFS). METHODS: Clinical, PET/CT and ctDNA data of DLBCL patients who underwent peripheral blood ctDNA testing and corresponding PET/CT scans during the same period were retrospectively analyzed. At the time of ctDNA sampling and PET scan, patients were divided into baseline and relapsed/refractory (R/R) groups according to different disease conditions. CtDNA mutation abundance was expressed as variant allele frequency (VAF), including maximum VAF (maxVAF) and mean VAF (meanVAF). Total metabolic tumour volume (TMTV) and total lesion glycolysis (TLG) were obtained by the 41% maximum normalized uptake value method, and the distance between the two farthest lesions (Dmax) was used to assess the correlation between PET parameters and ctDNA mutation abundance using Spearman correlation analysis. The receiver operating characteristic (ROC) curves were used to obtain the optical cut-off values of those parameters in predicting PFS in the baseline and R/R groups, respectively. Survival curves were outlined using the Kaplan-Meier method and log-rank test was performed to compare survival differences. RESULTS: A total of 67 DLBCL patients ï¼»28 males and 39 females, median age 56.0(46.0, 67.0) yearsï¼½ were included and divided into baseline group (29 cases) and R/R group (38 cases). Among these PET parameters, baseline TMTV, TLG, and Dmax were significantly correlated with baseline ctDNA mutation abundance, except for maximum standardized uptake value (SUVmax) (maxVAF vs TMTV: r=0.711; maxVAF vs TLG: r=0.709; maxVAF vs Dmax: r=0.672; meanVAF vs TMTV: r=0.682; meanVAF vs TLG: r=0.677; meanVAF vs Dmax: r=0.646). While in all patients, these correlations became weaker significantly. Among R/R patients, only TMTV had a weak correlation with meanVAF (r=0.376). ROC analysis showed that, the specificity of TMTV, TLG and Dmax in predicting PFS was better than mutation abundance, while the sensitivity of ctDNA mutation abundance was better. Except R/R patients, TMTV, TLG, Dmax, and VAF were significantly different at normal/elevated lactate dehydrogenase in baseline group and all patients (all P<0.05). Survival curves indicated that high TMTV (>109.5 cm3), high TLG (>2 141.3), high Dmax (>33.1 cm) and high VAF (maxVAF>7.74%, meanVAF>4.39%) were risk factors for poor PFS in baseline patients, while only high VAF in R/R patients (both maxVAF and meanVAF >0.61%) was a risk factor for PFS. CONCLUSION: PET-derived parameters correlate well with ctDNA mutation abundance, especially in baseline patients. VAF of ctDNA predicts PFS more sensitively than PET metabolic parameters, while PET metabolic tumour burden with better specificity. TMTV, TLG and VAF all have good prognostic value for PFS. PET/CT combined with ctDNA has potential for further studies in prognostic assessment and personalized treatment.

IL-1R8 expression in DLBCL regulates NK cell recruitment and influences patient prognosis.

The precise biological function of Interleukin-1 receptor 8 (IL-1R8) in diffuse large B-cell lymphoma (DLBCL) is still not well understood. Our goal is to decipher the profile of IL-1R8 expression status in DLBCL and to explore how IL-1R8 is involved in DLBCL progression. Utilizing a tissue microarray consisting of 70 samples of DLBCL tumors alongside 15 samples of tonsillitis, our investigation revealed a parallel expression profile of IL-1R8 between the tumor tissues and tonsillitis samples (p > 0.05). Nevertheless, an intriguing association emerged, as heightened expression of IL-1R8 correlated significantly with unfavorable survival outcomes in patients with DLBCL (p < 0.05). The status of IL-1R8 expression did not directly regulate proliferation (p > 0.05) and apoptosis (p > 0.05) in DLBCL cells via CCK8 and apoptotic assays. Subsequent chemotaxis analysis indicated that natural killer (NK) cell recruitment could be suppressed by IL-1R8 signaling in DLBCL, at least partially through CXCL1 inhibition (p < 0.05). The status of IL-1R8 expression in tumor tissues exhibited a negative correlation with the density of CD57+ NK cell infiltration (p < 0.05), while it did not demonstrate a significant association with CD3+ T cells (p > 0.05), CD68+ macrophages (p > 0.05), or S-100+ dendritic cells (p > 0.05). In line with this observation, elevated levels of NK cell infiltration demonstrated a significant positive correlation with improved overall survival (OS) among patients diagnosed with DLBCL (p < 0.05). Our data suggests the immuno-regulating potential of IL-1R8 through NK cell recruitment in DLBCL, providing novel insights into future immuno-modulating therapies.

Significant increase in MIC-A and MIC-B and soluble MIC-A and MIC-B in canine lymphomas.

Non-Hodkin's lymphoma (NHL) is the most frequent hematologic malignancy in humans and dogs. NKG2D is one of the most critical receptors on NK cells, recognizing their natural ligands on malignant cells such as A and B major histocompatibility complex-related proteins (MIC-A and MIC-B). Soluble molecules (sMIC-A and sMIC-B) can interfere with immune synapsis between NK cells and tumor cells, impeding NK cytotoxicity. The main objectives of this study were to analyze, in dogs with diffuse large B cell lymphoma, NK cell lymphoma, and reactive lymphadenopathies, the role of NK cells, their activating receptors NKG2D and NKp46, and their ligands MIC-A and MIC-B, as well as soluble molecules sMIC-A and sMIC-B. Thirty-six dogs with a possible diagnosis of NHL and eight healthy dogs were studied. NHL was diagnosed in 28 (78 %) dogs; in the other 8 (22 %), reactive lymphadenopathies were present. Most of the lymphomas corresponded to B cell NHL (82 %). The most predominant subtype was diffuse large B cell lymphoma (21, 71.5 %), followed by five cases (18 %) that were Non-B Non-T lymphomas (presumably NK cell lymphomas) and other B cell lymphomas (3, 10.5%). There were no cases of T cell NHL. MIC-A was positive in 7 of 27 (26 %) cases of NHL, and MIC-B in 20 of 27 (74 %) NHL. In non-malignant lymphadenopathies, three (37.5 %) dogs were positive for MIC-A, and five (62.5 %) expressed MIC-B. Dogs with lymphoma had higher numbers of NK cells than eight healthy dogs. In 15 dogs (12 cases with NHL and three cases with reactive adenopathies) and eight controls, there were no differences in the number of NK cells expressing NKP46 and NKG2D. NHL dogs had higher values of sMIC-A and sMIC-B. B-cell and NK cell lymphomas correspond to 86 % and 14 % of all canine lymphomas. MIC-A, MIC-B, and sMIC-A and sMIC-B were increased in canine lymphomas.

Clinicopathological Characteristics and Prognosis of Diffuse Large B-Cell Lymphoma in Relation to CA-125 and CA 19-9 Expression.

Background: Some epithelial tumors express the carbohydrate antigen 125 (Cancer antigen-125, CA-125) and CA 19-9, especially ovarian and pancreatic tumors. Patients with non-Hodgkin lymphoma (NHL) were reported to have a close association between serum CA-125 levels and adverse prognostic factors with worse survival. We aimed to investigate CA-125 and 19-9 expression in nodal diffuse large B-cell lymphoma, not otherwise specified (DLBCL NOS) tissues using immunohistochemistry (IHC) and their relations to clinicopathological manifestations and patients' survival. Methods: 65 cases of DLBCL NOS were examined. A modified mechanical pencil tip was used to construct Manual Tissue Micro-array (TMA) blocks. Immunohistochemical staining for CA-125 and CA 19-9 was performed and scored semi-quantitatively. All relations were analyzed using established statistical methodologies. Results: Aberrant expression of CA 19-9 was detected in 12% of cases without any expression of CA-125. Moreover, 75% of the CA 19-9 positive cases were statistically significantly associated with anemia and performance status 1. Also, 75% of the CA 19-9 positive cases were females. Conclusions: CA 19-9 was aberrantly expressed in 12% of nodal DLBCL NOS cases and significantly related to anaemia and performance status but not to survival. In cases of DLBCL NOS, CA 19-9 expression cannot be considered an independent prognostic factor. CA-125 was not expressed in nodal DLBCL NOS tissues, necessitating re-evaluation studies. Therefore, it is advised to conduct more research to clarify the potential correlation between serum and tissue CA 19-9 levels and other clinic-pathological characteristics of nodal and extranodal DLBCL NOS patients.

Targeted Polymersome Delivery of a Stapled Peptide for Drugging the Tumor Protein p53:BCL-2-Family Axis in Diffuse Large B-Cell Lymphoma.

Diffuse large B-cell lymphoma (DLBCL) remains a formidable diagnosis in need of new treatment paradigms. In this work, we elucidated an opportunity for therapeutic synergy in DLBCL by reactivating tumor protein p53 with a stapled peptide, ATSP-7041, thereby priming cells for apoptosis and enhancing their sensitivity to BCL-2 family modulation with a BH3-mimetic, ABT-263 (navitoclax). While this combination was highly effective at activating apoptosis in DLBCL in vitro, it was highly toxic in vivo, resulting in a prohibitively narrow therapeutic window. We, therefore, developed a targeted nanomedicine delivery platform to maintain the therapeutic potency of this combination while minimizing its toxicity via packaging and targeted delivery of a stapled peptide. We developed a CD19-targeted polymersome using block copolymers of poly(ethylene glycol) disulfide linked to poly(propylene sulfide) (PEG-SS-PPS) for ATSP-7041 delivery into DLBCL cells. Intracellular delivery was optimized in vitro and validated in vivo by using an aggressive human DLBCL xenograft model. Targeted delivery of ATSP-7041 unlocked the ability to systemically cotreat with ABT-263, resulting in delayed tumor growth, prolonged survival, and no overt toxicity. This work demonstrates a proof-of-concept for antigen-specific targeting of polymersome nanomedicines, targeted delivery of a stapled peptide in vivo, and synergistic dual intrinsic apoptotic therapy against DLBCL via direct p53 reactivation and BCL-2 family modulation.

Leukemic presentation and progressive genomic alterations of MCD/C5 diffuse large B-cell lymphoma (DLBCL).

Diffuse large B-cell lymphoma (DLBCL) is a heterogenous group of lymphoid malignancies. Based on gene expression profiling, it has been subdivided into germinal center (GC)-derived and activated B-cell (ABC) types. Advances in molecular methodologies have further refined the subclassification of DLBCL, based on recurrent genetic abnormalities. Here, we describe a distinct case of DLBCL that presented in leukemic form. DNA sequencing targeting 275 genes revealed pathogenically relevant mutations of CD79B, MyD88, TP53, TBL1XR1, and PIM1 genes, indicating that this lymphoma would be best classified as MCD/C5 DLBCL, an ABC subtype. Despite an initial good clinical response to BTK inhibitor ibrutinib, anti-CD20 antibody rituxan, alkylating agent bendamustine, and hematopoietic stem-cell transplant, the lymphoma relapsed, accompanied by morphologic and molecular evidence of disease progression. Specifically, the recurrent tumor developed loss of TP53 heterozygosity (LOH) and additional chromosomal changes central to ABC DLBCL pathogenesis, such as PRDM1 loss. Acquired resistance to ibrutinib and rituxan was indicated by the emergence of BTK and FOXO1 mutations, respectively, as well as apparent activation of alternative cell-activation pathways, through copy-number alterations (CNAs), detected by high-resolution chromosomal microarrays. In vitro, studies of relapsed lymphoma cells confirmed resistance to standard BTK inhibitors but sensitivity to vecabrutinib, a noncovalent inhibitor active against both wild-type as well as mutated BTK. In summary, we provide in-depth molecular characterization of a de novo leukemic DLBCL and discuss mechanisms that may have contributed to the lymphoma establishment, progression, and development of drug resistance.

An artificial intelligence method using FDG PET to predict treatment outcome in diffuse large B cell lymphoma patients.

Convolutional neural networks (CNNs) may improve response prediction in diffuse large B-cell lymphoma (DLBCL). The aim of this study was to investigate the feasibility of a CNN using maximum intensity projection (MIP) images from 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET) baseline scans to predict the probability of time-to-progression (TTP) within 2 years and compare it with the International Prognostic Index (IPI), i.e. a clinically used score. 296 DLBCL 18F-FDG PET/CT baseline scans collected from a prospective clinical trial (HOVON-84) were analysed. Cross-validation was performed using coronal and sagittal MIPs. An external dataset (340 DLBCL patients) was used to validate the model. Association between the probabilities, metabolic tumour volume and Dmaxbulk was assessed. Probabilities for PET scans with synthetically removed tumors were also assessed. The CNN provided a 2-year TTP prediction with an area under the curve (AUC) of 0.74, outperforming the IPI-based model (AUC = 0.68). Furthermore, high probabilities (> 0.6) of the original MIPs were considerably decreased after removing the tumours (< 0.4, generally). These findings suggest that MIP-based CNNs are able to predict treatment outcome in DLBCL.

High BECN1 Expression Negatively Correlates with BCL2 Expression and Predicts Better Prognosis in Diffuse Large B-Cell Lymphoma: Role of Autophagy.

Diffuse large B-cell lymphoma (DLBCL) is characterized by high molecular and clinical heterogeneity. Autophagy, a lysosome-driven catabolic process devoted to macromolecular turnover, is fundamental in maintaining normal hematopoietic stem cells and progenitors homeostasis, and its dysregulation plays a critical role in the initiation and progression of hematological malignancies. One main regulator of autophagy is BECLIN-1, which may interact alternatively with either BCL-2, thus allowing apoptosis, or PI3KC3, thus promoting autophagy. The altered expression of BCL2 and BECN1 correlates with lymphoma outcomes, but whether this is associated with dysregulated cross-talk between autophagy and apoptosis remains to be elucidated. Analysis of the TCGA database revealed that BCL2 and BECN1 mRNA expression were inversely correlated in DLBCL patients. In representative DLBCL cell lines exposed to doxorubicin, the cells highly expressing BCL-2 were resistant, while the ones highly expressing BECLIN-1 were sensitive, and this correlated with low and high autophagy flux, respectively. Venetoclax targeting of BCL-2 increased while the spautin-1-mediated inhibition of BECLIN-1-dependent autophagy reversed doxorubicin sensitivity in the former and in the latter, respectively. By interrogating the TCGA DLBCL dataset, we found that BCL2 and BECN1 acted as negative and positive prognostic markers for DLBCL, respectively. The differentially expressed gene analysis in the respective cohorts revealed that BCL2 positively correlated with oncogenic pathways (e.g., glucose transport, HIF1A signaling, JAK-STAT signaling, PI3K-AKT-mTOR pathway) and negatively correlated with autophagy-related transcripts, while BECN1 showed the opposite trend. Notably, patients with high BECN1 expression displayed longer survival. Our data reveal, for the first time, that the modulation of BECLIN-1-dependent autophagy influences the prognosis of DLBCL patients and provide a mechanistic explanation supporting the therapeutic use of drugs that, by stimulating autophagy, can sensitize lymphoma cells to chemotherapy.

BCL-W makes only minor contributions to MYC-driven lymphoma development.

The BH3-mimetic drug Venetoclax, a specific inhibitor of anti-apoptotic BCL-2, has had clinical success for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. Attention has now shifted towards related pro-survival BCL-2 family members, hypothesising that new BH3-mimetic drugs targeting these proteins may emulate the success of Venetoclax. BH3-mimetics targeting pro-survival MCL-1 or BCL-XL have entered clinical trials, but managing on-target toxicities is challenging. While increasing evidence suggests BFL-1/A1 is a resistance factor for diverse chemotherapeutic agents and BH3-mimetic drugs in haematological malignancies, few studies have explored the role of BCL-W in the development, expansion, and therapeutic responses of cancer. Previously, we found that BCL-W was not required for the ongoing survival and growth of various established human Burkitt lymphoma and diffuse large B cell lymphoma cell lines. However, questions remained about whether BCL-W impacts lymphoma development. Here, we show that BCL-W appears dispensable for MYC-driven lymphomagenesis, and such tumours arising in the absence of BCL-W show no compensatory changes to BCL-2 family member expression, nor altered sensitivity to BH3-mimetic drugs. These results demonstrate that BCL-W does not play a major role in the development of MYC-driven lymphoma or the responses of these tumours to anti-cancer agents.

CARD11 gain of function upregulates BCL2A1 expression and promotes resistance to targeted therapies combination in B-cell lymphoma.

A strategy combining targeted therapies is effective in B-cell lymphomas (BCL), such as mantle cell lymphoma (MCL), but acquired resistances remain a recurrent issue. In this study, we performed integrative longitudinal genomic and single-cell RNA-sequencing analyses of patients with MCL who were treated with targeted therapies against CD20, BCL2, and Bruton tyrosine kinase (OAsIs trial). We revealed the emergence of subclones with a selective advantage against OAsIs combination in vivo and showed that resistant cells were characterized by B-cell receptor (BCR)-independent overexpression of NF-κB1 target genes, especially owing to CARD11 mutations. Functional studies demonstrated that CARD11 gain of function not only resulted in BCR independence but also directly increased the transcription of the antiapoptotic BCL2A1, leading to resistance against venetoclax and OAsIs combination. Based on the transcriptional profile of OAsIs-resistant subclones, we designed a 16-gene resistance signature that was also predictive for patients with MCL who were treated with conventional chemotherapy, underlying a common escape mechanism. Among druggable strategies to inhibit CARD11-dependent NF-κB1 transduction, we evaluated the selective inhibition of its essential partner MALT1. We demonstrated that MALT1 protease inhibition led to a reduction in the expression of genes involved in OAsIs resistance, including BCL2A1. Consequently, MALT1 inhibition induced synergistic cell death in combination with BCL2 inhibition, irrespective of CARD11 mutational status, both in vitro and in vivo. Taken together, our study identified mechanisms of resistance to targeted therapies and provided a novel strategy to overcome resistance in aggressive BCL. The OAsIs trial was registered at www.clinicaltrials.gov #NCT02558816.