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Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogeneous disease that requires personalized clinical treatment. Assigning patients to different risk categories and cytogenetic abnormality and genetic mutation groups has been widely applied for prognostic stratification of DLBCL. Increasing evidence has demonstrated that dysregulated metabolic processes contribute to the initiation and progression of DLBCL. Metabolic competition within the tumor microenvironment is also known to influence immune cell metabolism. However, metabolism- and immune-related stratification has not been established. Here, 1660 genes involved in 84 metabolic pathways were selected and tested to establish metabolic clusters (MECs) of DLBCL. MECs established based on independent lymphoma datasets distinguished different survival outcomes. The CIBERSORT algorithm and EcoTyper were applied to quantify the relative abundance of immune cell types and identify variation in cell states for 13 lineages comprising the tumor micro environment among different MECs, respectively. Functional characterization showed that MECs were an indicator of the immune microenvironment and correlated with distinctive mutational characteristics and oncogenic signaling pathways. The novel immune-related MECs exhibited promising clinical prognostic value and potential for informing DLBCL treatment decisions., (© 2024 John Wiley & Sons Ltd.)

OX40 enhances the T-cell activation via costimulatory signaling. However, its molecular characteristics and value in predicting response to immunochemotherapy in DLBCL remain largely unexplored. Here, we performed an integrative analysis of sequencing and multiplex immunofluorescence staining, and discovered abnormally higher expression of OX40 in DLBCL patients. Elevated OX40 could activate T cells leading to a higher immune score for tumor immune microenvironment (TiME). OX40 upregulation simultaneously happened with immune-related genes including PD-1, CTLA4 and TIGIT et,al. Patients with high OX40 expression exhibited a lower Ann Arbor stage and IPI score and more easily achieved a complete response/partial response. The analysis of infiltrated T-cell subset revealed that patients with a greater number of CD4+/OX40+ or CD8+/OX40+ T cells had a longer OS. Our findings indicated that OX40 shapes an inflamed tumor immune microenvironment and predicts response to immunochemotherapy, providing insights for the application of OX40 agonist in DLBCL patients., Competing Interests: Declaration of Competing Interest The authors declare that no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Secondary central nervous system (SCNS) involvement is an infrequent but universally fatal event in diffused large B-cell lymphoma. The occurrence rate of SCNS involvement is approximately 5% but comes with a poor prognosis ever after. However, existing risk models to predict the incidence and prognosis of these patients with SCNS involvement lack both efficiency and accuracy. Controversy has also been reported regarding which risk factor may best identify the population with a high CNS relapse rate. In this study, we retrospectively analyzed 831 patients with diffused large B-cell lymphoma, diagnosed between March 2008 and June 2018 in Tianjin Medical University Cancer Institute and Hospital, Beijing Cancer Hospital, and Cancer Hospital of The University of Chinese Academy of Science. Risk factors and nomogram were identified and established based on Fine and Gray's competing risk analysis. Among these patients, 55 (6.6%) of them eventually developed SCNS involvement. The 1- and 2-year incidence for SCNS involvement were 3.9% and 4.7%, respectively. The median time from de novo diagnosis to CNS relapse was 8 months, and the median overall survival of these patients was 28 months. Considering the competing mortality before SCNS involvement, Fine and Gray's competing risk model was performed to analyze the characteristics related to SCNS involvement, and identified risk factors as the multiple extranodal involvements, elevated LDH and AMC level, and the involvement of breast, adrenal gland/kidney, pulmonary and bone. Corresponding factors were integrated into the competing nomogram for SCNS involvement (c-index = 0.778). In conclusion, we present the first predictive nomogram to evaluate the risk to develop SCNS involvement in de novo DLBCL patients, which may help in both prognostic evaluation and clinical decision for this subgroup., (© 2021 John Wiley & Sons Ltd.)

Epigenetic modifications contribute to lymphomagenesis. Here, we performed an expression clustering analysis and identified two epigenetic-related clusters (EC1 and EC2). EC1 presented abundant TP53, MYD88, HIST1H1D, HIST1H1C, KMT2D and EZH2 mutations and an inferior prognosis. Pathways involved in the regulation of DNA methylation/demethylation, histone methyltransferase activity, and protein methyltransferase activity were significantly enriched in EC1. However, EC2 was frequently accompanied by B2M, CD70 and MEF2B mutations, which presented with enrichments in DNA damage repair, cytokine-mediated and B-cell activated immune signaling, increased levels of CD8 + T-, γδT- and T helper-cells, as well as immune scores and immunogenic cell death (ICD) modulators. According to the prediction, EC1 was more sensitive to vorinostat, serdemetan and navitoclax. However, ruxolitinib, cytarabine and CP466722 were more suitable treatments for EC2. The novel immune-related epigenetic signature exhibits promising clinical predictive value for diffuse large B-cell lymphoma (DLBCL), particularly for guiding epigenetic therapeutic regimens. R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) based combination treatment regimens are suggested., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

TP53 mutations correlate with inferior survival in many cancers. APR-246 is a compound to shift mutant p53 and exhibits anti-cancer effects. Among its effects, APR-246 facilitates the binding of restored p53 mutants to target genes and their transcription. A set of 2464 DLBCL cases from multiple cohorts including our center, was integrated to identify the type and localization of TP53 mutations and clinical impacts. APR-246 was applied in TP53-mutated DLBCL cells and xenograft mouse models to explore the anti-tumor effect. TP53 mutations frequency was 16% and TP53 mutations correlated with poor overall survival (OS) and progression-free survival (PFS) in all cases, especially in germinal center B-cell-like (GCB) and unclassified (UNC) subtypes. Notably, TP53 single mutations in the DNA binding domain (DBD) led to poor OS and PFS. Specifically, mutations in exon 7 correlated with poorer OS, while mutations in exons 5 and 6 associated with inferior PFS. APR-246 induces p53-dependent ferritinophagy of DLBCL cells with TP53 missense mutation on exon 7 and ferroptosis of DLBCL cells harboring wild-type TP53 and other TP53 mutations. TP53 mutations on exons 5, 6 and 7 are predictors of progression and survival. Targeting mutant p53 by APR-246 is a promising therapeutic approach for DLBCL patients., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Concurrent follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) (defined as FL/DLBCL) have been considered an important pathological feature in cell lymphoma. However, clinicopathological information and prognostic factors in these cases are scarce. The aim of this study was to construct a prediction index to compare with traditional prognostic models., Methods: Retrospectively enrolled, previously untreated FL/DLBCL ( n = 121) patients, as well as those with pure FL 1-3a ( n = 471), were assessed. De novo DLBCL ( n = 529) were used as controls. Kaplan-Meier curves were plotted to compare the outcomes among the three groups. Multivariate analysis identified risk factors associated with overall survival (OS) in FL/DLBCL patients. A clinicopathological prognosis index (CPPI) was developed to predict OS based on the Cox proportional hazards model., Results: The outcomes of FL/DLBCL patients were intermediate between pure FL 1-3a and de novo DLBCL patients, with a 5-year PFS of 70%, 59%, and 48% ( P < 0.05) and 5-year OS of 80%, 70% and 60% ( P < 0.05), respectively. Cox regression analysis showed that the prognostic factors of OS for FL/DLBCL patients included FL grade, cell of origin, and Ann Arbor stage. A nomogram and clinicopathological prognostic index (CPPI) were developed to predict the OS for FL/DLBCL patients based on these factors. The area under the curve (AUC) of the CPPI for 3- and 5-year OS prediction was 0.782 and 0.860, respectively. This was superior to that of the International Prognostic Index (IPI), Follicular Lymphoma International Prognostic Index (FLIPI), and FLIPI2 in the 0.540-0.819 ( P < 0.01) range., Conclusions: A valid OS estimation in FL/DLBCL patients, using the recommended CPPI, may be useful in routine clinical practice., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2022 Zhenjie Qu et al.)

Background: Targeting the PD-1/PD-L1/L2 (programmed cell death protein 1/programmed cell death ligand 1/ligand 2) pathway combined with other immunosuppressive signalings, such as CD73/A2aR (A2a adenosine receptor) adenosine signaling, has emerged as a promising strategy for cancer treatment. The genetic characteristics of these immune checkpoints need to be further investigated in diffuse large B-cell lymphoma (DLBCL)., Methods: We performed whole-exome sequencing/targeted deep sequencing to investigate the genetic characteristics of PD-1/PD-L1/L2 and CD73/A2aR. The immunosuppressive effect of these two pathways on the tumor microenvironment was evaluated via RNA sequencing. Single-cell RNA sequencing was further applied to investigate the dysfunctional CD8+ T cells. In addition, multiplex immunofluorescence staining was used to quantitatively assess the expression of dysfunctional CD8 + T cells in DLBCL., Results: SP140 was identified as a novel translocation partner for PD-L1, and a new inversion was detected between PD-L1 and PD-L2, both leading to the upregulation of PD-L1 expression. CD73 genetic mutations did not increase mRNA and protein expression. Patients with genetically altered CD73 tended to have a better overall survival than patients with wild-type CD73. Both PD-1/PD-L1 and CD73/A2aR signaling mediated the immunosuppressive microenvironment in DLBCL. The numbers of CD8 + T cells with PD-1 and A2aR expression were positively correlated with the number of dysfunctional CD8 + T cells (R 2 =0.974, p = 0.013). According to the grades of dysfunctional CD8 + T cells we defined, grade 1 dysfunctional CD8 + T cells, with either PD-1 + or A2aR + , were significantly associated with poorer survival than grade 0 dysfunctional CD8 + T cells, with both PD-1 - and A2aR - ; and patients with grade 2 dysfunctional CD8 + T cells showed the worst clinical outcomes., Conclusions: This study describes the additional genetic basis of PD-L1 overexpression and characterizes certain genetic alterations of CD73/A2aR in DLBCL. The degree of T-cell dysfunction is correlated with clinical outcomes. Strategies that reverse T-cell dysfunction by inhibiting PD-1/PD-L1/L2, particularly in combination with CD73/A2aR, may show potential as effective therapeutic options for DLBCL., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogenous malignancy, early identification of patients for relapse remains challenging. The potential to non-invasively monitor tumour evolutionary dynamics of DLBCL needs to be further established. In the present study, 17 tumour biopsy and 38 plasma samples from 38 patients with high-intermediate/high-risk DLBCL were evaluated at baseline. Longitudinal blood samples were also collected during therapy. Circulating tumour DNA (ctDNA) was analysed using targeted sequencing based on a gene panel via a recently developed methodology, circulating single-molecule amplification and re-sequencing technology (cSMART). We found that the most frequently mutated genes were tumour protein p53 (TP53; 42·1%), histone-lysine N-methyltransferase 2D (KMT2D; 28·9%), caspase recruitment domain family member 11 (CARD11; 21·1%), cAMP response element-binding protein binding protein (CREBBP; 15·8%), β 2 -microglobulin (B2M; 15·8%), and tumour necrosis factor alpha-induced protein 3 (TNFAIP3; 15·8%). The mutation profiles between ctDNA and matched tumour tissue showed good concordance; however, more mutation sites were detected in ctDNA samples. Either TP53 or B2M mutations before treatment predicted poor prognosis. Analysis of dynamic blood samples confirmed the utility of ctDNA for the real-time assessment of treatment response and revealed that the increases in ctDNA levels and changes in KMT2D mutation status could be useful predictors of disease progression. Our present results suggest that ctDNA is a promising method for the detection of mutation spectrum and serves as a biomarker for disease monitoring and predicting clinical recurrence., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Diffuse large B-cell lymphoma (DLBCL) presents a great clinical challenge and has a poor prognosis, with immune-related genes playing a crucial role. We aimed to develop an immune-related prognostic signature for improving prognosis prediction in DLBCL. Samples from the GSE31312 dataset were randomly allocated to discovery and internal validation cohorts. Univariate Cox, random forest, LASSO regression and multivariate Cox analyses were utilized to develop a prognostic signature, which was verified in the internal validation cohort, entire validation cohort and external validation cohort (GSE10846). The tumor microenvironment was investigated using the CIBERSORT and ESTIMATE tools. Gene set enrichment analysis (GSEA) was further applied to analyze the entire GSE31312 cohort. We identified four immune-related genes (CD48, IL1RL, PSDM3, RXFP3) significantly associated with overall survival. Based on discovery and validation cohort analyses, this four-gene signature could classify patients into high- and low-risk groups, with significantly different prognoses. Activated memory CD4 T cells and activated dendritic cells were significantly decreased in the high-risk group, and these patients had lower immune scores. GSEA revealed enrichment of signaling pathways, such as T cell receptor, antigen receptor-mediated, antigen processing and presentation of peptide antigen via MHC class I, in the low-risk group. In conclusion, a robust signature based on four immune-related genes was successfully constructed for predicting prognosis in DLBCL patients.

This study aimed to identify the prognostic factors in patients with Waldeyer's ring diffuse large B-cell lymphoma (WR-DLBCL), comparing the efficacy of radiotherapy (RT) for the WR-DLBCL patients in the pre-rituximab and rituximab eras. We conducted a retrospective analysis of 134 patients diagnosed with WR-DLBCL. Univariate and multivariate analyses were performed to identify the prognostic factors for WR-DLBCL. Then, we divided these patients into the rituximab plus chemotherapy group (R-chemotherapy) (n = 88) and chemotherapy group (n = 46), and the Kaplan-Meier and Cox regression model analyses were applied to investigate the treatment value of RT in both the groups. Multivariate analysis revealed international prognostic index (IPI) ≥ 3 and chemotherapy without rituximab as significant risk factors for the progression-free survival (PFS, IPI ≥ 3: p = 0.001; chemotherapy without rituximab: p = 0.002) and overall survival (OS, IPI ≥ 3, p < 0.001; chemotherapy without rituximab, p = 0.024). Rituximab combined with chemotherapy significantly improved PFS (p = 0.002) and OS (p = 0.006) in these patients. RT did not significantly contribute to the survival in the overall cohort analysis, whereas in the subgroup analysis, RT significantly improved the PFS (p = 0.025) and OS (p = 0.029) for the patients in the chemotherapy group, but not in the R-chemotherapy group. In conclusion, the WR-DLBCL patients could benefit from RT in the pre-rituximab era, whereas the addition of rituximab to chemotherapy significantly improved the survival of WR-DLBCL patients, and the clinical benefit of RT was reduced., (© 2021 John Wiley & Sons Ltd.)

Background: The purpose of this study was to construct a new typing model for diffuse large B-cell lymphoma (DLBCL) patients based on the B-cell receptor (BCR) and explore its potential molecular mechanism., Methods: BCR repertoire sequencing and whole-exome sequencing were performed on formalin-fixed paraffin-embedded samples from 12 DLBCL patients. Subsequently, a typing model was built with cluster analysis, and prognostic indicators between the two groups were compared to verify the typing model. Then, mutation and bioinformatics analyses were conducted to investigate the potential biomarkers of prognostic differences between the two groups., Results: Based on BCR sequencing data, we divided patients into two clusters (cluster 1 and cluster 2); this classification differed from the traditional typing method (GCB and non-GCB), in which cluster 1 included some non-GCB patients. The progression-free survival (PFS), overall survival (OS), metastasis and Shannon diversity index of IGH V-J and survival after chemotherapy were significantly different (P < 0.05) between the two clusters, but no statistical significance was found between the GCB and non-GCB groups. The mutation status of 248 genes was significantly different between cluster 1 and cluster 2. Among them, FTSJ3, MAGED2, and ODF3L2 were the specific mutated genes in all patients in cluster 2, and these genes could be considered critical to the different prognoses of the two clusters of DLBCL patients., Conclusion: We constructed a new typing model of DLBCL based on BCR repertoire sequencing that can better predict the survival time after chemotherapy. FTSJ3, MAGED2, and ODF3L2 may represent key genes for the difference in prognosis between the two clusters.

Tim-3 is a promising target for antitumor immunotherapy. A number of clinical trials are evaluating the efficacy of anti-Tim-3 therapies as a single agent or combinations in solid tumors and haematologic malignancies. However, there remains a considerable lack of data on Tim-3 signalling, especially the genetic characteristics and immune microenvironment, in diffuse large B cell lymphoma (DLBCL). Herein, we identified three genetic mutations in galectin-9, a major ligand of Tim-3, in six patients with DLBCL (6/188, 3.2%) that were not detected in the COSMIC database. The Oncomine database showed that the mRNA levels of Tim-3 were higher in DLBCL cells than those in normal B cells. Multiplexed immunofluorescence revealed that patients with Tim-3-expressing tumor-infiltrating lymphocytes (Tim-3 + TILs) exhibited poor outcomes than those with Tim-3 - TILs ( p = 0.041). The median survival times of these patients were 65.0 (95% confidence interval (CI): 71.2-88.6) and 79.9 months (95% CI: 54.4-75.6), respectively. Furthermore, we defined a novel subtype of exhausted T cells, named as exhausted Tim-3 + CD8 + T cells, and found that patients with exhausted Tim-3 + CD8 + T cells (median survival, 62.8 months, 95% CI: 50.0-75.6) exhibited shorter survival than those with nonexhausted Tim-3 - CD8 + T cells (median survival, 82.5 months, 95% CI: 72.0-92.9; p = 0.034). Overall, these findings provide the genetic status of the Tim-3 ligand in DLBCL. Patients with Tim-3 + TILs and exhausted Tim-3 + CD8 + T cells exhibited inferior survival, thus highlighting the possibility of potential therapeutic applications of the inhibition of Tim-3 alone or in combination with other immune checkpoints for treatment of patients with DLBCL., Competing Interests: The authors declare no competing interests., (Copyright © 2020 Tingting Zhang et al.)

De novo CD5-positive diffuse large B-cell lymphoma (CD5+ DLBCL) is increasingly recognized as a distinct pathologic phenomenon with a specific clinical picture. However, CD5+ DLBCL has not been studied on a large scale in China. In this study, we show that CD5+ DLBCL occurs at a low frequency (9.2%). Comparison of clinical characteristics of CD5+ vs CD5- DLBCL showed that CD5+ DLBCL was more frequently elderly (>60 years) and had B symptoms, high-performance status, stage III-IV, an IPI score >2 and bone marrow involvement. Patients with CD5+ DLBCL had tumours with a higher prevalence of BCL-2 and p53 overexpression than CD5- DLBCL. Patients with CD5+ DLBCL had inferior progression-free survival (PFS) and overall survival (OS) than did patients with CD5- DLBCL. For CD5+ DLBCL, the patients who were treated with rituximab showed significantly better PFS and OS than those treated without rituximab. However, patients treated with RCHOP showed similar PFS and OS when compared with the group treated with intensive therapy. In addition, patients with p53 and CD5 co-expression had the worst PFS and OS. In conclusion, CD5+ DLBCL was associated with unfavorable clinicopathologic variables and with inferior survival. CD5+ DLBCL has a high frequency of p53 overexpression, and CD5 augments the negative effect of p53 overexpression in DLBCL., (© 2019 John Wiley & Sons, Ltd.)

Novel immune checkpoint blockades, including those targeting CD73 and A2aR, are being evaluated in malignancies in clinical trials. Here, we investigated the expression of CD73 and A2aR as well as tumor-infiltrating lymphocytes (TILs), and analyzed their correlations with clinicopathological characteristics and survival in diffuse large B-cell lymphoma (DLBCL). We found that CD73 expression on tumor cells, rather than the total protein and gene levels of CD73, was associated with survival. Patients with CD73 + /Pax-5 + (median survival, 57.8 months; 95% CI, 46.4-69.3) experienced significantly poorer outcomes than those with CD73 - /Pax-5 + (median survival, 73.5 months; 95% CI, 65.9-81.2). Additionally, A2aR expression on both total TILs and CD8 + TILs was correlated with survival. Patients with A2aR + TILs (median survival, 53.3 months; 95% CI, 40.6-66.0) had a significantly shorter survival time than patients with A2aR - TILs (median survival, 74.5 months; 95% CI, 67.5-81.5). Spearman's rank test showed that CD73 expression on tumor cells was positively correlated with A2aR expression on TILs (R = 0.395, p = 0.001). We further found that patients could be more precisely stratified through the combination of CD73 tumor cell expression and A2aR TILs expression, and patients with CD73 + /Pax-5 + and A2aR + TILs experienced the worst outcome. We also revealed that patients with CD73 + /Pax-5 + and low CD8 + TILs or low absolute lymphocyte counts had unfavorable outcomes. Overall, our findings uncovered that patients with CD73 + on tumor cells as well as A2aR + on TILs or low CD8 + TILs exhibited inferior survival, supporting potential combination strategies using CD73/A2aR immunosuppressive blockades as treatment options for DLBCL patients., (© 2019 UICC.)

Background: Double-expression lymphoma (DEL) is a rare subgroup of diffuse large B-cell lymphoma (DLBCL), which has coexpression of MYC and BCL-2. Coexpression of MYC and BCL-2 is considered a prognostic marker portending poor outcomes. However, the prognostic effect of BCL-2 and BCL-6 expression in DLBCL remains controversial., Materials and Methods: Immunohistochemical staining was performed to detect MYC, BCL-2 and BCL-6 expression in 212 patients with newly diagnosed DLBCL and assess the prognostic effects of BCL-2 and BCL-6 expression. The DLBCL patients were treated with R-CHOP (rituximab plus cyclophosphamide, doxorubicin, vincristine [Oncovin], prednisone)-like regimens., Results: Retrospective analysis revealed that BCL-2 + and BCL-2 + /MYC + were prognostic factors indicative of poor outcomes. Patients with BCL-2 + and/or MYC + expression had a poorer prognosis than that of patients with BCL-2 - and/or MYC - expression. Patients with BCL-2 + /MYC - expression showed a trend toward poorer survival than those with BCL-2 - /MYC + expression, suggesting that BCL-2 plays a more important role than MYC. Also, patients with BCL-6 - /MYC + expression had poorer progression-free survival than those with BCL-6 + /MYC + expression. In addition, patients with BCL-2 + /MYC + /BCL-6 - expression had the worst prognosis, suggesting that BCL-6 - is a prognostic factor for poor outcomes for MYC + DLBCL patients. Altogether, our findings have shown that BCL-2 is an independent prognostic factor and possibly plays a more important role than MYC in MYC + DLBCL patients. Furthermore, we found that BCL-6 - expression could also be a prognostic factor portending poor outcomes for MYC + DLBCL patients., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Programmed death-1 (PD-1) /programmed death-ligand 1 (PD-L1) engagement usually leads to diminished antitumor T-cell responses, which mediates the immune escape of tumor cells. However, little is known whether PD-1/PD-L1 could directly activates intracellular oncogenic signaling pathways in tumor cells. The purpose of this study is to investigate whether intracellular AKT/mTOR signaling could be directly activated by PD-1/PD-L1 during the malignant progression in diffuse large B-cell lymphoma (DLBCL). Detection of the expression of PD-L1 and p-AKT by immunohistochemistry (IHC) showed that both proteins were overexpressed in 54% and 48% DLBCL cases, respectively. Spearman test showed that PD-L1 expression was correlated with p-AKT expression (R=0.244, χ2=5.962; P=0.017) and the expression of PD-L1 and p-AKT were also correlated with clinic-pathological characteristics. In addition, survival analysis showed that DLBCL patients who co-expressed PD-L1 and p-AKT had significantly poorer outcome than patients with single positive or both negative expression (P<0.05). In vitro, total PD-L1 and membrane PD-L1 (mPD-L1) proteins were overexpressed in five DLBCL cell lines by western blot and flow cytometry. We observed that AKT/mTOR pathway was activated in DLBCL cells after stimulated with human recombination PD-1/Fc. Taken together, these results suggested that the combination of PD-1/PD-L1 antibodies and AKT/mTOR inhibitor might be a promising and novel therapeutic approach for DLBCL in the future., Competing Interests: The authors declared no conflicts of interest.