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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., (Copyright © 2024 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Double expressor lymphoma (DEL) is a subset of diffuse large B-cell lymphoma (DLBCL) characterized by the co-expression of MYC and BCL2 proteins with a poor prognosis. However, there are no standard criteria for evaluating the morphologic features of DEL. We aimed to analyze the prognostic value of the starry-sky pattern (SSP) and its correlation with clinicopathologic and genetic features in 153 DEL cases. The SSP was significantly associated with aggressive parameters, including c-MYC overexpression, CD5 expression, higher IPI, and age-adjusted IPI. In the univariate survival analyses, the presence of SSP was associated with unfavorable progression-free survival (PFS) (p = 0.040), and tended towards an adverse overall survival (OS) (p = 0.061). However, when c-MYC was overexpressed, SSP was significantly correlated with inferior OS (p = 0.019). In the multivariate survival analysis, SSP was also associated with poor PFS (p = 0.048). Additionally, next-generation sequencing data revealed SSP was significantly associated with the KMT2D mutation and had different genetic mutation profiles from non-SSP. In conclusion, SSP may represent morphologic characteristics of aggressiveness in DEL., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The clinical significance of extra copy (EC) genotypes of BCL2 , MYC , and BCL6 have not been fully elucidated. We evaluated the EC and translocation statuses of BCL2 , MYC , and BCL6 in 190 diffuse large B-cell lymphoma (DLBCL) cases using fluorescence in situ hybridization. EC genotype was sub-classified according to copy number-gained tumor cell ratio (EC1, >20% but ≤50%; EC2, >50%). Only the BCL2 -EC groups, not MYC -EC or BCL6 -EC groups, displayed significantly increased immunoreactivity of the corresponding protein. Moreover, the BCL2 -EC2 group was significantly associated with poor overall survival (OS) and progression-free survival (PFS) in a 147 R-CHOP-treated patient subset, which was also statistically significant as per the multivariate survival analysis for PFS. No significant differences in the survival of MYC , BCL6 , concurrent BCL2/MYC , BCL6/MYC , BCL2/BCL6, or triple EC groups were observed. BCL2 -EC may contribute to increased BCL-2 protein expression and serve as a predictor of treatment outcomes in DLBCL.

Overexpression of the BCL2 protein has been reported as a poor prognostic factor for diffuse large B-cell lymphoma (DLBCL). However, there are currently no standardized criteria for evaluating BCL2 protein expression. We aimed to evaluate the prognostic value of BCL2 expression determined by immunohistochemistry (IHC), incorporating both the staining intensity and proportion, in patients with de novo DLBCL who received rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) as first-line treatment. We defined tumors with BCL2 expression in nearly all tumor cells with a uniformly strong intensity by IHC as BCL2 super-expressor. The BCL2 super-expressors (n = 35) showed significantly worse event-free survival (EFS; HR, 1.903; 95% CI, 1.159-3.126, P = 0.011) and overall survival (OS; HR, 2.467; 95% CI, 1.474-4.127, P = 0.001) compared with the non-BCL2 super-expressors (n = 234) independent of the international prognostic index (IPI), cell of origin (COO), and double expressor status in the training set (n = 269). The adverse prognostic impact of BCL2 super-expression was confirmed in the validation set (n = 195). When the survival outcomes were evaluated in the entire cohort (n = 464), BCL2 super-expressor group was significantly associated with inferior EFS and OS regardless of IPI, COO, MYC expression, and stages. BCL2 super-expressors had genetic aberrations enriched in the NOTCH and TP53 signaling pathways. This study suggests that the BCL2 super-expressor characterizes a distinct subset of DLBCL with a poor prognosis and warrants further investigation as a target population for BCL-2 inhibitors., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Diffuse large B-cell lymphoma (DLBCL) is a fatal heterogenous neoplasm. Recent clinical trials have failed partly due to nebulous criteria for defining high-risk patients. Patients with double-expresser lymphoma (DEL) have a poor prognosis and are resistant to conventional treatment. However, many diagnostic and clinical controversies still surround DEL partly due to the arbitrariness of criteria for the diagnosis of DEL. In this study, we suggest a refined method for diagnosing DEL by evaluating the concurrent expression of BCL2 and MYC at the single-cell level (dual-protein-expressing lymphoma [DUEL]). For the proof of concept, a multiplex immunofluorescence assay for CD20, BCL2, and MYC was performed and quantitatively analyzed using spectral image analysis in patients. The analysis results and clinical applicability were verified by using dual-color immunohistochemistry performed on 353 independent multicenter patients who had been uniformly treated with standard therapy. DUEL showed significantly worse overall survival (OS) and event-free survival (EFS) (P=0.00011 and 0.00035, respectively). DUEL status remained an independent adverse prognostic variable with respect to the International Prognostic Index risk and the cell of origin. Moreover, the advantage of determining DUEL status by dual-color immunohistochemistry was shown by more robust classification and more homogeneous high-risk subgroup patient identification in both training (n=271) (OS: P<0.0001; EFS: P<0.0001) and validation sets (n=82) (OS: P=0.0087; EFS: P<0.0001). This concept of DUEL is more consistent with carcinogenesis and has greater practical utility, hence it may provide a better basis for both basic and clinical research for the development of new therapeutics., Competing Interests: Conflicts of Interest and Source of Funding: Supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2021R1C1C2007816 to J.R. and NRF-2020R1A2C2006362 to C.-S.P.) and Asan Institute for Life Sciences, Seoul, Korea (no. 2017-0432 to D.H.L.). The authors have disclosed that they have no significant relationships with, or financial interest in, any commercial companies pertaining to this article., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Multiple studies have demonstrated that diffuse large B-cell lymphoma (DLBCL) can be divided into subgroups based on their biology; however, these biological subgroups overlap clinically. Using machine learning, we developed an approach to stratify patients with DLBCL into four subgroups based on survival characteristics. This approach uses data from the targeted transcriptome to predict these survival subgroups. Using the expression levels of 180 genes, our model reliably predicted the four survival subgroups and was validated using independent groups of patients. Multivariate analysis showed that this patient stratification strategy encompasses various biological characteristics of DLBCL, and only TP53 mutations remained an independent prognostic biomarker. This novel approach for stratifying patients with DLBCL, based on the clinical outcome of rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone therapy, can be used to identify patients who may not respond well to these types of therapy, but would otherwise benefit from alternative therapy and clinical trials., (© 2022. The Author(s).)

Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma with high mutation burdens but a low response rate to immune checkpoint inhibitors. In this study, we performed targeted next-generation sequencing and fluorescent multiplex immunohistochemistry, and investigated the clinical significance and immunological effect of mutation numbers in 424 DLBCL patients treated with standard immunochemotherapy. We found that KMT2D and TP53 nonsynonymous mutations (MUT) were significantly associated with increased nonsynonymous mutation numbers, and that high mutation numbers (MUT high ) were associated with significantly poorer clinical outcome in germinal center B-cell-like DLBCL with wild-type TP53 . To understand the underlying mechanisms, we identified a gene-expression profiling signature and the association of MUT high with decreased T cells in DLBCL patients with wild-type TP53 . On the other hand, in overall cohort, MUT high was associated with lower PD-1 expression in T cells and PD-L1 expression in macrophages, suggesting a positive role of MUT high in immune responses. Analysis in a whole-exome sequencing dataset of 304 patients deposited by Chapuy et al. validated the correlation of MUT- KMT2D with genomic complexity and the significantly poorer survival associated with higher numbers of genomic single nucleotide variants in activated B-cell-like DLBCL with wild-type TP53 . Together, these results suggest that KMT2D inactivation or epigenetic dysregulation has a role in driving DLBCL genomic instability, and that genomic complexity has adverse impact on clinical outcome in DLBCL patients with wild-type TP53 treated with standard immunochemotherapy. The oncoimmune data in this study have important implications for biomarker and therapeutic studies in DLBCL., Competing Interests: HN, MLN, and QA are employees of NeoGenomics Laboratories, Inc.. MA is an employee of Genomic Testing Corporative, LCA. Other authors declare no conflicts of interest., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Despite central nervous system (CNS) relapse occurring in >10% of high-risk diffuse large B-cell lymphoma (DLBCL) patients, the role of CNS-directed prophylaxis is controversial in the absence of randomized controlled trials. In this retrospective study, we aimed to evaluate the safety and efficacy of prophylactic high-dose methotrexate (HD-MTX) on CNS relapse and survival outcomes in 258 newly diagnosed R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone)-treated high-risk DLBCL patients, based on the initial treatment intent (ITT) of the physician on the use of prophylactic HD-MTX. Patients were classified into an ITT HD-MTX group (n = 128) and a non-ITT HD-MTX group (n = 130). The CNS relapse rate was not significantly different between these groups, with 2-year CNS relapse rates of 12.4% and 13.9%, respectively (P = 0.96). Three-year progression-free survival and overall survival rates in the ITT HD-MTX and non-ITT HD-MTX groups were 62.4% vs 64.5% (P = 0.94) and 71.7% vs 71.4% (P = 0.7), respectively. Also, propensity score-matched analyses showed no significant differences in the time-to-CNS-relapse, progression-free survival, or overall survival. The ITT HD-MTX group showed a higher incidence of grade ≥ 3 oral mucositis and elevated alanine aminotransferase. Prophylactic HD-MTX does not improve CNS relapse rate or survival outcomes in high-risk DLBCL patients, and it is accompanied by increased toxicities., (© 2021 by The American Society of Hematology.)

The role of the Epstein-Barr virus (EBV) status in the blood for predicting survival in post-transplantation lymphoproliferative disorders-diffuse large B-cell lymphoma (PTLD-DLBCL) is unknown. We evaluated the prognostic values of pre-treatment EBV-encoded small RNA (EBER) detected with in situ hybridization in tissues and EBV DNA in the whole blood (WB) and plasma in 58 patients with monomorphic PTLD-DLBCL after solid organ transplantation. There were no significant differences in the rates of overall response, complete response, and survival according to EBER EBV and WB EBV status. In contrast, patients with positive plasma EBV DNA had significantly lower rates of overall response (60.0% vs. 94.4%, P = 0.043) and complete response (40.0% vs. 88.9%, P = 0.019) as well as worse progression-free survival (PFS) (P = 0.035) and overall survival (OS) (P = 0.039) compared with patients with negative plasma EBV DNA. In multivariate analysis, plasma EBV DNA positivity was a significantly unfavorable prognostic factor for PFS [hazard ratio (HR) 4.92, 95% confidence interval (CI) 1.22-19.86, P = 0.025] and OS (HR 4.48, 95% CI 1.14-17.63, P = 0.032). Despite small number of 6 patients with plasma EBV positivity, plasma EBV DNA positivity might be more prognostic for survival than EBER or WB EBV DNA positivity in patients with monomorphic PTLD-DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is the major type of aggressive B-cell lymphoma. High-grade B-cell lymphoma (HGBCL) with MYC / BCL2 double-hit (DH) represents a distinct entity with dismal prognosis after standard immunochemotherapy in the current WHO lymphoma classification. However, whether TP53 mutation synergizes with MYC abnormalities ( MYC rearrangement and/or Myc protein overexpression) contributing to HGBCL-like biology and prognosis is not well investigated. In this study, patients with DLBCL with MYC/TP53 abnormalities demonstrated poor clinical outcome, high-grade morphology, and distinct gene expression signatures. To identify more effective therapies for this distinctive DLBCL subset, novel MYC/TP53/BCL-2-targeted agents were investigated in DLBCL cells with MYC/TP53 dual alterations or HGBCL- MYC / BCL2 -DH. A BET inhibitor INCB057643 effectively inhibited cell viability and induced apoptosis in DLBCL/HGBCL cells regardless of MYC / BCL2 / TP53 status. Combining INCB057643 with a MDM2-p53 inhibitor DS3032b significantly enhanced the cytotoxic effects in HGBCL-DH without TP53 mutation, while combining with the BCL-2 inhibitor venetoclax displayed potent therapeutic synergy in DLBCL/HGBCL cells with and without concurrent TP53 mutation. Reverse-phase protein arrays revealed the synergistic molecular actions by INCB057643, DS3032b and venetoclax to induce cell-cycle arrest and apoptosis and to inhibit AKT/MEK/ERK/mTOR pathways, as well as potential drug resistance mechanisms mediated by upregulation of Mcl-1 and RAS/RAF/MEK/ERK pathways. In summary, these findings support subclassification of DLBCL/HGBCL with dual MYC/TP53 alterations, which demonstrates distinct pathobiologic features and dismal survival with standard therapy, therefore requiring additional targeted therapies. IMPLICATIONS: The clinical and pharmacologic studies suggest recognizing DLBCL with concomitant TP53 mutation and MYC abnormalities as a distinctive entity necessary for precision oncology practice. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/19/2/249/F1.large.jpg., (©2020 American Association for Cancer Research.)

Purpose: In contrast to the Western diffuse large B-cell lymphoma (DLBCL), prognostic impact of age in a Korean population with DLBCL has not been fully evaluated., Materials and Methods: Six hundred and eight DLBCL patients treated with rituximab-containing chemotherapeutic regimens from January 2002 to March 2012 in Asan Medical Center were enrolled. Survival models using the restricted cubic spine-transformed age variable were constructed to evaluate non-linear relationships between age and survival outcome. Finally, age was categorized according to the conventional international prognostic index (IPI), National Comprehensive Cancer Network (NCCN)-IPI, and Grupo Español de Linfomas/Trasplante Autólogo de Médula Ósea (GELTAMO)-IPI schemes and the prognostic implications were evaluated., Results: The relative hazard did not change significantly during the first to fifth decades, but began to increase exponentially in patients aged over 62 years. This pattern or relationship was also retained in a multivariate model fitted to the age-adjusted IPI and relative dose intensity. Multivariate survival analysis revealed that age > 75 years, but not age > 60 years, was associated independently with poor overall and progression-free survival when the relative dose intensity and age-adjusted IPI were taken into account., Conclusion: The outcome of DLBCL in Korean populations may deteriorate rapidly as age exceeds 62 years. Therefore, a consensus cutoff value for age in Korean DLBCL patients should be determined to better predict prognosis.

The standard of treatment for completely resected limited-stage diffuse large B cell lymphoma (DLBCL) in patients without residual lesions has not yet been established. Previously, we designed a phase II trial to evaluate the safety and efficacy of three cycles of abbreviated R-CHOP in patients with completely resected limited-stage DLBCL and reported favorable survival outcomes. We present the long-term follow-up results to taking into account the importance of delayed relapse in patients with limited-stage DLBCL. With a median follow-up duration of 62.7 months (range, 60.2-75.5 months), the 5-year OS and DFS rates were both 95.0% (95% confidence interval, 85.59-104.11%). Only one patient experienced disease progression which was confirmed at 12.3 months, and one patient with primary intestinal DLBCL developed non-small cell lung cancer 6 years after treatment. The long-term results of our data support the use of three cycles of abbreviated R-CHOP for patients with completely resected limited-stage DLBCL. The study was reviewed and approved by the review boards of the participating institutes and registered at ClinicalTrials.gov , number NCT01279902, in August 3, 2010.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous entity of B-cell lymphoma. Cell-of-origin (COO) classification of DLBCL is required in routine practice by the World Health Organization classification for biological and therapeutic insights. Genetic subtypes uncovered recently are based on distinct genetic alterations in DLBCL, which are different from the COO subtypes defined by gene expression signatures of normal B cells retained in DLBCL. We hypothesize that classifiers incorporating both genome-wide gene-expression and pathogenetic variables can improve the therapeutic significance of DLBCL classification. To develop such refined classifiers, we performed targeted RNA sequencing (RNA-Seq) with a commercially available next-generation sequencing (NGS) platform in a large cohort of 418 DLBCLs. Genetic and transcriptional data obtained by RNA-Seq in a single run were explored by state-of-the-art artificial intelligence (AI) to develop a NGS-COO classifier for COO assignment and NGS survival models for clinical outcome prediction. The NGS-COO model built through applying AI in the training set was robust, showing high concordance with COO classification by either Affymetrix GeneChip microarray or the NanoString Lymph2Cx assay in 2 validation sets. Although the NGS-COO model was not trained for clinical outcome, the activated B-cell-like compared with the germinal-center B-cell-like subtype had significantly poorer survival. The NGS survival models stratified 30% high-risk patients in the validation set with poor survival as in the training set. These results demonstrate that targeted RNA-Seq coupled with AI deep learning techniques provides reproducible, efficient, and affordable assays for clinical application. The clinical grade assays and NGS models integrating both genetic and transcriptional factors developed in this study may eventually support precision medicine in DLBCL., (© 2020 by The American Society of Hematology.)

We evaluated real-world effectiveness and safety of CT-P10 (Truxima ® ) compared with originator rituximab in diffuse large B-cell lymphoma (DLBCL) treatment. Before and after the introduction of CT-P10 to our institute (November 2017), 221 newly-diagnosed DLBCL patients received rituximab with standard cyclophosphamide, vincristine, doxorubicin and prednisone. Patients received originator rituximab throughout ( n  = 95), switched from originator rituximab to CT-P10 ( n  = 36), or received CT-P10 throughout ( n  = 90). There were no significant differences between groups in overall response rate (91.6% vs 94.4% vs 96.7%, respectively; p  = 0.403) or complete response rate (84.2% vs 77.8% vs 86.7%, respectively; p  = 0.467). Kaplan-Meier survival curves also showed no significant differences in progression-free survival and overall survival between groups (log-rank p  = 0.794 and p  = 0.955, respectively). Safety profiles were comparable between treatment groups. These data support the ability of CT-P10 to successfully replace originator rituximab in DLBCL treatment and, given the lowered financial barrier, to improve the overall prognosis for DLBCL patients.

BCL2 overexpression has been reported to be associated with poor prognosis in patients with diffuse large B-cell lymphoma (DLBCL). However, currently there is no consensus on the evaluation of BCL2 expression and only the proportion of BCL2 positive cells are evaluated for the determination of BCL2 positivity. This study aimed to define BCL2 positivity by quantitative analysis integrating both the intensity and proportion of BCL2 expression. BCL2 expression of 332 patients (221 patients for the training set and 111 patients for the validation set) with newly diagnosed DLBCL who received R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) were analyzed using the tumor-specific automated quantitative analysis (AQUA) scoring method based on multiplex immunofluorescence. In the training set, high BCL2 AQUA score (N = 86, 38.9%) was significantly associated with poor prognosis (p = 0.01, HR 2.00; 95% CI [1.15-3.49]) independent of international prognostic index, cell of origin, and MYC expression. The poor prognostic impact of the high BCL2 AQUA score was validated in the validation set. AQUA scoring of BCL2 expression incorporating both the intensity and proportion of BCL2 positive cells was independently associated with survival outcomes of patients with primary DLBCL treated with R-CHOP.

The distinct clinical characteristics of patients at diagnosis of diffuse large B-cell lymphoma (DLBCL), who experience a late relapse compared with those who relapse early, require reappraisal in the era of R-CHOP. Data from the lymphoma registry of the Asan Medical Center between 2002 and 2015 were retrospectively reviewed. Among 846 DLBCL patients treated with first-line R-CHOP and achieved complete response, 169 (20%) relapsed, occurring late (>2 years) in 51 (6%). Compared with early relapses, late relapses were associated with a lower International Prognostic Index score ( p =.001), lower incidence of B-symptoms ( p =.004), lower lactate dehydrogenase ( p =.001), lower stage (I-II; p =.001), and the median overall survival from relapse was significantly longer (2.4 vs. 1.0 years, p =.001). Patients with a late relapse who responded to salvage chemotherapy and received autologous stem-cell transplantation, had higher 5-year survival rate (55.4% vs. 20.2%). Time of relapse and LDH level were independent prognostic factors affecting survival.

Background: The upregulated expression of the JAK/STAT pathway promotes tumor growth in Hodgkin lymphoma (HL) and primary mediastinal large B-cell lymphoma (PMBCL). Based on the hypothesis that JAK2 is a therapeutic target, we performed a prospective pilot study using ruxolitinib., Methods: Relapsed or refractory patients with HL or PMBCL were eligible for this study, and JAK2 amplification was assessed by fluorescence in situ hybridization. Ruxolitinib was administered orally at a dose of 20 mg twice daily for a 28-day cycle. Treatment was continued for up to 16 cycles or until progressive disease or intolerability. The primary objective was to assess the overall disease control rate comprising complete response (CR), partial response (PR), or stable disease (SD)., Results: We analyzed 13 HL patients and six PMBCL patients. All responders (one CR, five PR, and one SD) had HL whereas all cases of PMBCL progressed after first or second cycle. The disease control rate for HL was 54% (7/13) with median response duration of 5.6 months. JAK2 amplification was present in six of nine patients tested (four HL, two PMBCL), and three of these HL patients showed PR (n = 2) or SD (n = 1). None of the three HL patients shown to not have JAK2 amplification responded to ruxolitinib. Most treatment-related adverse events were grade 1 or 2 and manageable., Conclusions: Ruxolitinib has single-agent activity against HL but does not act against PMBCL with or without JAK2 amplification., Trial Registration: The study population was patients who had relapsed or refractory HL or PMBCL, and patients were registered for our pilot study after providing written informed consent between November 2013 and November 2015 (CilinicalTrials.gov: NCT01965119).

Background: Diffuse large B-cell lymphoma (DLBCL) harbors somatic hypermutation (SHM) in the immunoglobulin heavy chain and light chain variable region genes, IGHV and IGK/LV. Recent studies have revealed that IGV SHM creates neoantigens that activate T-cell responses against B-cell lymphoma., Methods: To determine the clinical relevance of IGV SHM in DLBCL treated with standard immunochemotherapy, we performed next-generation sequencing of the immunoglobulin variable regions and complementarity determining region 3 (CDR3) for 378 patients with de novo DLBCL. The prognostic effects of IGV SHM and ongoing SHM or intra-clonal heterogeneity were analyzed in the training (192 patients), validation (186 patients), and overall DLBCL cohorts. To gain mechanistic insight, we analyzed the predicted IG-derived neoantigens' immunogenicity potential, determined by the major histocompatibility complex-binding affinity and the frequency-of-occurrence of T cell-exposed motifs (TCEMs) in a TCEM repertoire derived from human proteome, microbiome, and pathogen databases. Furthermore, IGV SHM was correlated with molecular characteristics of DLBCL and PD-1/L1 expression in the tumor microenvironment assessed by fluorescent multiplex immunohistochemistry., Results: SHM was commonly found in IGHV and less frequently in IGK/LV. High levels of clonal IGHV SHM (SHM high ) were associated with prolonged overall survival in DLBCL patients, particularly those without BCL2 or MYC translocation. In contrast, long heavy chain CDR3 length, the presence of IGHV ongoing SHM in DLBCL, and high clonal IGK/LV SHM in germinal center B-cell-like (GCB)-DLBCL were associated with poor prognosis. These prognostic effects were significant in both the training and validation sets. By prediction, the SHM high groups harbored more potentially immune-stimulatory neoantigens with high binding affinity and rare TCEMs. PD-1/L1 expression in CD8 + T cells was significantly lower in IGHV SHM high than in SHM low patients with activated B-cell-like DLBCL, whereas PD-1 expression in CD4 + T cells and PD-L1 expression in natural killer cells were higher in IGK/LV SHM high than in SHM low patients with GCB-DLBCL. PD-L1/L2 (9p24.1) amplification was associated with high IGHV SHM and ongoing SHM., Conclusions: These results show for the first time that IGV SHM high and ongoing SHM have prognostic effects in DLBCL and potential implications for PD-1/PD-L1 blockade and neoantigen-based immunotherapies.

We evaluated the efficacy of systemic high-dose methotrexate (HD-MTX) for CNS prophylaxis in a prospectively recruited cohort of DLBCL patients at high risk of CNS relapse. High-risk CNS relapse was defined as the involvement of ≥ 2 extranodal sites with elevated lactate dehydrogenase (LDH); CNS international prognostic index (CNS-IPI) ≥ 4; or involvement of high-risk extranodal sites, including bone marrow, breasts, testes, and paranasal sinuses. Overall, 130 patients who received at least two cycles of standard chemoimmunotherapy were evaluated; of these, 64 patients received additional systemic HD-MTX, either on day 15 of alternating cycles or 2-5 weeks after completion of primary therapy. Patients receiving HD-MTX showed a generally higher risk of CNS relapse than the other 66 patients not receiving prophylaxis. The estimated 2-year cumulative CNS relapse, progression-free survival and overall survival rates were 8.1%, 66.3%, and 77.5%, respectively, in patients who received systemic HD-MTX and 6.9%, 67.4%, and 71.4%, respectively, in patients without prophylaxis, with no significant between-group differences. Although this study does not show that systemic HD-MTX is definitively effective, these results may be of help to other researchers investigating the utility of HD-MTX prophylaxis in mitigating negative risk factors.

Programmed cell death protein 1/programmed cell death protein ligand1 (PD-1/PD-L1) interaction is an important immune checkpoint targeted by anti-PD-1/PD-L1 immunotherapies. However, the observed prognostic significance of PD-1/PD-L1 expression in diffuse large B-cell lymphoma treated with the standard of care has been inconsistent and even contradictory. To clarify the prognostic role of PD-1/PD-L1 expression and interaction in diffuse large B-cell lymphoma, in this study we used 3-marker fluorescent multiplex immunohistochemistry and Automated Quantitative Analysis Technology to assess the CD3 + , PD-L1 + , and PD-1 + CD3 + expression in diagnostic samples and PD-1/PD-L1 interaction as indicated by presence of PD-1 + CD3 + cells in the vicinity of PD-L1 + cells, analyzed their prognostic effects in 414 patients with de novo diffuse large B-cell lymphoma, and examined whether PD-1/PD-L1 interaction is required for the prognostic role of PD-1 + /PD-L1 + expression. We found that low T-cell tissue cellularity, tissue PD-L1 + expression (irrespective of cell types), PD-1 + CD3 + expression, and PD-1/PD-L1 interaction showed hierarchical adverse prognostic effects in the study cohort. PD-1/PD-L1 interaction showed higher sensitivity and specificity than PD-1 + and PD-L1 + expression in predicting inferior prognosis in patients with high CD3 + tissue cellularity ("hot"/inflammatory tumors). However, both PD-1 + and PD-L1 + expression showed adverse prognostic effects independent of PD-1/PD-L1 interaction, and PD-1/PD-L1 interaction showed favorable prognostic effect in PD-L1 + patients without high CD3 + tissue cellularity. Macrophage function and tumor-cell MYC expression may contribute to the PD-1-independent adverse prognostic effect of PD-L1 + expression. In summary, low T-cell tissue cellularity has unfavorable prognostic impact in diffuse large B-cell lymphoma, and tissue PD-L1 + expression and T-cell-derived PD-1 + expression have significant adverse impact only in patients with high T-cell infiltration. PD-1/PD-L1 interaction in tissue is essential but not always responsible for the inhibitory effect of PD-L1 + /PD-1 + expression. These results suggest the benefit of PD-1/PD-L1 blockade therapies only in patients with sufficient T-cell infiltration, and the potential of immunofluorescent assays and Automated Quantitative Analysis in the clinical assessment of PD-1/PD-L1 expression and interaction.

Cell-of-origin (COO) classification of diffuse large B cell lymphoma (DLBCL) is increasingly important due to its prognostic significance and the development of subtype-specific therapeutics. We compared the clinical utility of the Lymph2Cx assay against four widely used immunohistochemical algorithms in 150 R-CHOP-treated DLBCL patients using archival tissue. In contrast to the predominance of germinal center B cell-like (GCB) subtype in Western populations, Lymph2Cx assay classified more than half of the Korean cases as the activated B cell-like (ABC) subtype (ABC, 83/150 [55.3%]; GCB, 51/150 [34.0%]; unclassifiable, 16/150 [10.7%]). Predominance of ABC subtype tended to be more pronounced in the nodal lymphomas than in the extranodal lymphomas. However, among the primary extranodal sites, ABC subgroups predominated in primary testicular, breast, and adrenal gland lymphomas. The classification of COO by Lymph2Cx assay did not show any significant association with clinical parameters. The overall concordance rates of the immunohistochemical algorithms with the Lymph2Cx ranged from 78.0 to 84.3%. However, 47.1-66.7% of the cases of the Lymph2Cx-defined GCB subgroup were misclassified as the non-GCB class by the IHC algorithms. The survival of Lymph2Cx-classified COO subtypes was not significantly different in the present cohort. In conclusion, ABC subtype predominated over GCB in Korean patients. There are significant discrepancies between the immunohistochemistry and Lymph2Cx classifications, especially in GCB subtype.

The prognostic significances of the germinal center B-cell-like (GCB) and non-germinal center B-cell-like (non-GCB) types of diffuse large B-cell lymphoma (DLBCL) have been reported to be different. We analyzed the effect of the cell of origin (COO) of bone marrow (BM) involvement in patients with DLBCL who were treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) in a single institute.The subtype of BM involvement was evaluated in 633 patients who were diagnosed with primary DLBCL and had been treated with R-CHOP. BM trephine biopsies were analyzed, and immunohistochemical staining of CD20, CD79a, and CD3 was performed. Additional staining of CD10, Bcl-6, and MUM1 was performed to determine the COO based on a previously reported algorithm.BM involvement was present in 81 patients (12.8%). Among them, 30 patients (37.0%) had GCB-type BM involvement and 51 (63.0%) showed non-GCB-type involvement. Kaplan-Meier survival analysis showed that the non-GCB type had the worst progression-free survival (PFS) and overall survival (OS) (P <.001). In multivariate analysis controlled for the International Prognostic Index (IPI) score, non-GCB type was an independent predictor of PFS (P <.004) and OS (P =.042), whereas GCB type was not a prognostic factor independent of the IPI score.Further prognostication based on the COO of BM involvement is a useful indicator of PFS, independent of IPI score. Accurate staging based on the COO should be included in the examination of BM in DLBCL.

PTEN loss has been associated with poorer prognosis in many solid tumors. However, such investigation in lymphomas is limited. In this study, PTEN cytoplasmic and nuclear expression, PTEN gene deletion, and PTEN mutations were evaluated in two independent cohorts of diffuse large B-cell lymphoma (DLBCL). Cytoplasmic PTEN expression was found in approximately 67% of total 747 DLBCL cases, more frequently in the activated B-cell-like subtype. Nuclear PTEN expression was less frequent and at lower levels, which significantly correlated with higher PTEN mRNA expression. Remarkably, loss of PTEN protein expression was associated with poorer survival only in DLBCL with AKT hyperactivation. In contrast, high PTEN expression was associated with Myc expression and poorer survival in cases without abnormal AKT activation. Genetic and epigenetic mechanisms for loss of PTEN expression were investigated. PTEN deletions (mostly heterozygous) were detected in 11.3% of DLBCL, and showed opposite prognostic effects in patients with AKT hyperactivation and in MYC rearranged DLBCL patients. PTEN mutations, detected in 10.6% of patients, were associated with upregulation of genes involved in central nervous system function, metabolism, and AKT/mTOR signaling regulation. Loss of PTEN cytoplasmic expression was also associated with TP53 mutations, higher PTEN-targeting microRNA expression, and lower PD-L1 expression. Remarkably, low PTEN mRNA expression was associated with down-regulation of a group of genes involved in immune responses and B-cell development/differentiation, and poorer survival in DLBCL independent of AKT activation. Collectively, multi-levels of PTEN abnormalities and dysregulation may play important roles in PTEN expression and loss, and that loss of PTEN tumor-suppressor function contributes to the poor survival of DLBCL patients with AKT hyperactivation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

PRDM1/BLIMP-1, a master regulator of plasma-cell differentiation, is frequently inactivated in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) patients. Little is known about its genetic aberrations and relevant clinical implications. A large series of patients with de novo DLBCL was effectively evaluated for PRDM1/BLIMP-1 deletion, mutation, and protein expression. BLIMP-1 expression was frequently associated with the ABC phenotype and plasmablastic morphologic subtype of DLBCL, yet 63% of the ABC-DLBCL patients were negative for BLIMP-1 protein expression. In these patients, loss of BLIMP-1 was associated with Myc overexpression and decreased expression of p53 pathway molecules. In addition, homozygous PRDM1 deletions and PRDM1 mutations within exons 1 and 2, which encode for domains crucial for transcriptional repression, were found to show a poor prognostic impact in patients with ABC-DLBCL but not in those with germinal center B-cell-like DLBCL (GCB-DLBCL). Gene expression profiling revealed that loss of PRDM1/BLIMP-1 expression correlated with a decreased plasma-cell differentiation signature and upregulation of genes involved in B-cell receptor signaling and tumor-cell proliferation. In conclusion, these results provide novel clinical and biological insight into the tumor-suppressive role of PRDM1/BLIMP-1 in ABC-DLBCL patients and suggest that loss of PRDM1/BLIMP-1 function contributes to the overall poor prognosis of ABC-DLBCL patients.

Background: The appropriate number of chemotherapy cycles for limited stage diffuse large B-cell lymphoma (DLBCL) patients without gross residual lesions after complete resection, has not been specifically questioned. We performed a multicenter, single-arm, phase 2 study to investigate the feasibility of 3 cycles of abbreviated R-CHOP chemotherapy in low-risk patients with completely resected localized CD20+ DLBCL., Results: Between December 2010 and May 2013, we recruited 23 patients. One was excluded due to ineligibility, and hence, 22 were included in the final analysis. The primary sites comprised the intestine (n = 15), cervical lymph nodes (n = 4), stomach (n = 1), tonsil (n = 1), and spleen (n = 1). All patients successfully completed the 3 cycles of planned R-CHOP chemotherapy. Over a median follow-up of 39.5 months (95% confidence interval, 29.9-47.1 months), both the estimated 2-year disease-free survival and overall survival rates was 95% confidence interval, 85.9-104.1%. Only one patient with an international prognostic index of 2 experienced relapse and died. The most common grade 3 or 4 toxicity condition included neutropenia (n = 8, 36.4%). Three patients experienced grade 3 febrile neutropenia, but no grade 3 or 4 non-hematologic toxicity was observed., Materials and Methods: DLBCL patients without residual lesions after resection were enrolled and R-CHOP chemotherapy was repeated at 3-week-intervals over 3 cycles. The primary endpoint was 2-year disease-free survival., Conclusions: Three cycles of abbreviated R-CHOP immunochemotherapy is feasible for completely resected low risk localized DLBCL.

CD37 (tetraspanin TSPAN26) is a B-cell surface antigen widely expressed on mature B cells. CD37 is involved in immune regulation and tumor suppression but its function has not been fully elucidated. We assessed CD37 expression in de novo diffuse large B-cell lymphoma (DLBCL), and investigated its clinical and biologic significance in 773 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and 231 patients treated with CHOP. We found that CD37 loss (CD37 - ) in ∼60% of DLBCL patients showed significantly decreased survival after R-CHOP treatment, independent of the International Prognostic Index (IPI), germinal center B-cell-like (GCB)/activated B-cell-like (ABC) cell of origin, nodal/extranodal primary origin, and the prognostic factors associated with CD37 - , including TP53 mutation, NF-κB high , Myc high , phosphorylated STAT3 high , survivin high , p63 - , and BCL6 translocation. CD37 positivity predicted superior survival, abolishing the prognostic impact of high IPI and above biomarkers in GCB-DLBCL but not in ABC-DLBCL. Combining risk scores for CD37 - status and ABC cell of origin with the IPI, defined as molecularly adjusted IPI for R-CHOP (M-IPI-R), or IPI plus immunohistochemistry (IHC; IPI+IHC) for CD37, Myc, and Bcl-2, significantly improved risk prediction over IPI alone. Gene expression profiling suggested that decreased CD20 and increased PD-1 levels in CD37 - DLBCL, ICOSLG upregulation in CD37 + GCB-DLBCL, and CD37 functions during R-CHOP treatment underlie the pivotal role of CD37 status in clinical outcomes. In conclusion, CD37 is a critical determinant of R-CHOP outcome in DLBCL especially in GCB-DLBCL, representing its importance for optimal rituximab action and sustained immune responses. The combined molecular and clinical prognostic indices, M-IPI-R and IPI+IHC, have remarkable predictive values in R-CHOP-treated DLBCL., (© 2016 by The American Society of Hematology.)

It has been well established that nuclear factor kappa-B (NF-κB) activation is important for tumor cell growth and survival. RelA/p65 and p50 are the most common NF-kB subunits and involved in the classical NF-kB pathway. However, the prognostic and biological significance of RelA/p65 is equivocal in the field. In this study, we assessed RelA/p65 nuclear expression by immunohistochemistry in 487 patients with de novo diffuse large B-cell lymphoma (DLBCL), and studied the effects of molecular and pharmacological inhibition of NF-kB on cell viability. We found RelA/p65 nuclear expression, without associations with other apparent genetic or phenotypic abnormalities, had unfavorable prognostic impact in patients with stage I/II DLBCL. Gene expression profiling analysis suggested immune dysregulation and antiapoptosis may be relevant for the poorer prognosis associated with p65 hyperactivation in germinal center B-cell-like (GCB) DLBCL and in activated B-cell-like (ABC) DLBCL, respectively. We knocked down individual NF-κB subunits in representative DLBCL cells in vitro, and found targeting p65 was more effective than targeting other NF-κB subunits in inhibiting cell growth and survival. In summary, RelA/p65 nuclear overexpression correlates with significant poor survival in early-stage DLBCL patients, and therapeutic targeting RelA/p65 is effective in inhibiting proliferation and survival of DLBCL with NF-κB hyperactivation., Competing Interests: KHY receives research support from Roche Molecular System, Gilead Sciences Pharmaceutical, Seattle Genetics, Dai Sanyo Pharmaceutical, Adaptive Biotechnology, Incyte Pharmaceutical, and HTG Molecular Diagnostics. EDH is a Consultant for HTG Molecular Diagnostics. BMP joins speakers Burea for Celgene and Amgen, and a Consultant for Celgene.

The prognostic value of serum beta-2 microglobulin for diffuse large B-cell lymphoma (DLBCL) is not well known in the rituximab era. A retrospective registry data analysis of 833 patients with de novo DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) was conducted to establish the prognostic significance of serum beta-2 microglobulin at a ≥2.5 mg/L cutoff. Five-year progression-free survival (PFS, 76.1% vs. 41.0%; p < 0.001) and overall survival (OS, 83.8% vs. 49.2%; p < 0.001) were significantly worse in patients with elevated serum beta-2 microglobulin (n = 290, 34.8%). Furthermore, the five parameters of the International Prognostic Index, accompanying B symptoms, bone marrow involvement and impaired renal function were associated with worse PFS and OS. In multivariate analysis, elevated beta-2 microglobulin was a significant poor prognostic factor for PFS (hazard ratio [HR], 1.70; 95% confidence interval [CI], 1.29-2.24; p < 0.001) and OS (HR, 2.0; 95% CI, 1.47-2.75; p < 0.001). In an independent validation cohort of 258 R-CHOP treated patients with de novo DLBCL, elevated beta-2 microglobulin levels remained a significant poor prognostic factor for PFS (HR, 2.03; 95% CI, 1.23-3.32; p = 0.005) and exhibited a strong trend of association with worse OS (HR, 1.64; 95% CI, 0.98-2.75; p = 0.062). The significance of serum beta-2 microglobulin levels as an independent prognostic factor for patients with DLBCL receiving R-CHOP is confirmed.

Extranodal involvement is a well-known prognostic factor in patients with diffuse large B-cell lymphomas (DLBCL). Nevertheless, the prognostic impact of the extranodal scoring system included in the conventional international prognostic index (IPI) has been questioned in an era where rituximab treatment has become widespread. We investigated the prognostic impacts of individual sites of extranodal involvement in 761 patients with DLBCL who received rituximab-based chemoimmunotherapy. Subsequently, we established a new extranodal scoring system based on extranodal sites, showing significant prognostic correlation, and compared this system with conventional scoring systems, such as the IPI and the National Comprehensive Cancer Network-IPI (NCCN-IPI). An internal validation procedure, using bootstrapped samples, was also performed for both univariate and multivariate models. Using multivariate analysis with a backward variable selection, we found nine extranodal sites (the liver, lung, spleen, central nervous system, bone marrow, kidney, skin, adrenal glands, and peritoneum) that remained significant for use in the final model. Our newly established extranodal scoring system, based on these sites, was better correlated with patient survival than standard scoring systems, such as the IPI and the NCCN-IPI. Internal validation by bootstrapping demonstrated an improvement in model performance of our modified extranodal scoring system. Our new extranodal scoring system, based on the prognostically relevant sites, may improve the performance of conventional prognostic models of DLBCL in the rituximab era and warrants further external validation using large study populations.

Purpose: MYC is a critical driver oncogene in many cancers, and its deregulation in the forms of translocation and overexpression has been implicated in lymphomagenesis and progression of diffuse large B-cell lymphoma (DLBCL). The MYC mutational profile and its roles in DLBCL are unknown. This study aims to determine the spectrum of MYC mutations in a large group of patients with DLBCL, and to evaluate the clinical significance of MYC mutations in patients with DLBCL treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) immunochemotherapy., Experimental Design: We identified MYC mutations in 750 patients with DLBCL using Sanger sequencing and evaluated the prognostic significance in 602 R-CHOP-treated patients., Results: The frequency of MYC mutations was 33.3% at the DNA level (mutations in either the coding sequence or the untranslated regions) and 16.1% at the protein level (nonsynonymous mutations). Most of the nonsynonymous mutations correlated with better survival outcomes; in contrast, T58 and F138 mutations (which were associated with MYC rearrangements), as well as several mutations occurred at the 3' untranslated region, correlated with significantly worse survival outcomes. However, these mutations occurred infrequently (only in approximately 2% of DLBCL). A germline SNP encoding the Myc-N11S variant (observed in 6.5% of the study cohort) was associated with significantly better patient survival, and resulted in reduced tumorigenecity in mouse xenografts., Conclusions: Various types of MYC gene mutations are present in DLBCL and show different impact on Myc function and clinical outcomes. Unlike MYC gene translocations and overexpression, most MYC gene mutations may not have a role in driving lymphomagenesis. Clin Cancer Res; 22(14); 3593-605. ©2016 AACR., (©2016 American Association for Cancer Research.)

Primary testicular diffuse large B-cell lymphoma (PT-DLBCL) is a unique subtype of DLBCL. The impact of rituximab on survival and patterns of treatment failure in PT-DLBCL patient remain controversial. We analyzed the clinical and biological feature of 280 PT-DLBCL cases, 64% of which were treated with rituximab-containing regimens. Although most (95%) patients achieved complete remission, a continuous risk of relapse was observed. Rituximab significantly reduced the cumulative risk of relapse (P=0.022) and improved both progression-free survival (PFS, P=0.012) and overall survival (OS, P=0.027) of PT-DLBCL patients (5-year PFS, 56% vs 36%; 5-year OS, 68% vs 48%). Central nervous system and contralateral testis were the most common sites of relapse, but other extranodal and nodal sites of relapse were also observed. Most cases of PT-DLBCL had a non-germinal center B-cell like (84%) immunophenotype and an activated B-cell like (86%) gene expression profile (GEP) subtype. The distinctive GEP signature of primary testicular lymphoma was relevant to tumor cell proliferation, dysregulated expression of adhesion molecules and immune response, likely accounting for the poor outcome. Accordingly, forkhead box P1 transcription factor (FOXP1) and T-cell leukemia/lymphoma 1 (TCL1) oncogenic activation were confirmed and predicted a significant trend of poor survival. This study provides valuable observations for better understanding of both clinical and biological features in PT-DLBCL patients.

Diffuse large B-cell lymphoma (DLBCL) can be separated into 2 groups: nodal and extranodal disease. The aim of this study was to analyse the clinical features of skin lesions and survival outcomes of cutaneous DLBCL according to the primary tumour site. A total of 44 patients with cutaneous DLBCL were classified as primary cutaneous DLBCL, leg type or cutaneous DLBCL secondary to primary disease. Although skin lesion characteristics did not differ significantly between groups, extensive cutaneous lesions were more often observed in secondary cutaneous DLBCL compared with DLBCL, leg type. Secondary cutaneous DLBCL was more commonly associated with an advanced stage and higher International Prognostic Index score than DLBCL, leg type. DLBCL, leg type demonstrated a better survival outcome than secondary cutaneous DLBCL. The multiplicity of skin lesions and time-point of cutaneous involvement were associated with prognosis in secondary cutaneous DLBCL. Survival outcomes and prognostic factors differ depending on the primary tumour site of cutaneous DLBCL.

The role of p53 family member p63 in oncogenesis is the subject of controversy. Limited research has been done on the clinical implications of p63 expression in diffuse large B-cell lymphoma (DLBCL). In this study, we assessed p63 expression in de novo DLBCL samples (n=795) by immunohistochemistry with a pan-p63-monoclonal antibody and correlated it with other clinicopathologic factors and clinical outcomes. p63 expression was observed in 42.5% of DLBCL, did not correlate with p53 levels, but correlated with p21, MDM2, p16INK4A, Ki-67, Bcl-6, IRF4/MUM-1 and CD30 expression, REL gains, and BCL6 translocation. p63 was an independent favorable prognostic factor in DLBCL, which was most significant in patients with International Prognostic Index (IPI) >2, and in activated-B-cell-like DLBCL patients with wide- type TP53. The prognostic impact in germinal-center-B-cell-like DLBCL was not apparent, which was likely due to the association of p63 expression with high-risk IPI, and potential presence of ∆Np63 isoform in TP63 rearranged patients (a mere speculation). Gene expression profiling suggested that p63 has both overlapping and distinct functions compared with p53, and that p63 and mutated p53 antagonize each other. In summary, p63 has p53-like and p53-independent functions and favorable prognostic impact, however this protective effect can be abolished by TP53 mutations.

Double-hit B-cell lymphoma is a common designation for a group of tumors characterized by concurrent translocations of MYC and BCL2, BCL6, or other genes. The prognosis of concurrent MYC and BCL6 translocations is not well known. In this study, we assessed rearrangements and expression of MYC, BCL2 and BCL6 in 898 patients with de novo diffuse large B-cell lymphoma treated with standard chemotherapy (cyclophosphamide, doxorubicin, vincristine, and prednisone plus rituximab). Neither BCL6 translocation alone (more frequent in activated B-cell like diffuse large B-cell lymphoma) nor in combination with MYC translocation (observed in 2.0% of diffuse large B-cell lymphoma) predicted poorer survival in diffuse large B-cell lymphoma patients. Diffuse large B-cell lymphoma patients with MYC/BCL6 co-expression did have significantly poorer survival, however, MYC/BCL6 co-expression had no effect on prognosis in the absence of MYC/BCL2 co-expression, and had no additive impact in MYC+/BCL2+ cases. The isolated MYC+/BCL6+/BCL2- subset, more frequent in germinal center B-cell like diffuse large B-cell lymphoma, had significantly better survival compared with the isolated MYC+/BCL2+/BCL6- subset (more frequent in activated B-cell like diffuse large B-cell lymphoma). In summary, diffuse large B-cell lymphoma patients with either MYC/BCL6 rearrangements or MYC/BCL6 co-expression did not always have poorer prognosis; MYC expression levels should be evaluated simultaneously; and double-hit B-cell lymphoma needs to be refined based on the specific genetic abnormalities present in these tumors.

MYC dysregulation, including MYC gene rearrangement and Myc protein overexpression, is of increasing clinical importance in diffuse large B-cell lymphoma (DLBCL). However, the roles of MYC and the relative importance of rearrangement vs overexpression remain to be refined. Gaining knowledge about the tumor biology associated with MYC dysregulation is important to understand the roles of MYC and MYC-associated biology in lymphomagenesis. In this study, we determined MYC rearrangement status (n=344) and Myc expression (n=535) in a well-characterized DLBCL cohort, individually assessed the clinical and pathobiological features of patients with MYC rearrangement and Myc protein overexpression, and analyzed the prognosis and gene expression profiling signatures associated with these MYC abnormalities in germinal center B-cell-like and activated B-cell-like DLBCL. Our results showed that the prognostic importance of MYC rearrangement vs Myc overexpression is significantly different in germinal center B-cell-like vs activated B-cell-like DLBCL. In germinal center B-cell-like DLBCL, MYC-rearranged germinal center B-cell-like DLBCL patients with Myc overexpression significantly contributed to the clinical, biological, and prognostic characteristics of the overall Myc-overexpressing germinal center B-cell-like DLBCL group. In contrast, in activated B-cell-like DLBCL, the occurrence, clinical and biological features, and prognosis of Myc overexpression were independent of MYC rearrangement. High Myc levels and Myc-independent mechanisms, either tumor cell intrinsic or related to tumor microenvironment, conferred significantly worse survival to MYC-rearranged germinal center B-cell-like DLBCL patients, even among Myc(high)Bcl-2(high) DLBCL patients. This study provides new insight into the tumor biology and prognostic effects associated with MYC dysregulation and suggest that detection of both MYC translocations and evaluation of Myc and Bcl-2 expression is necessary to predict the prognosis of DLBCL patients.

Obesity was recently reported to confer a survival advantage in diffuse large B cell lymphoma (DLBCL) among Western populations. Given ethnic differences, previous studies recommended a revision of the WHO classification of obesity for Asians. We investigated the prognostic impact of body mass index (BMI) using modified WHO criteria in a retrospective cohort of 562 Korean patients with DLBCL. Patients were categorized into five groups according to BMI: 26 (4.6 %) as underweight (<18.5 kg/m(2)), 230 (40.9 %) as normal weight (18.5-22.9 kg/m(2)), 129 (23.0 %) as overweight (23.0-24.9 kg/m(2)), 160 (28.5 %) as obese (25.0-29.9 kg/m(2)), and 17 (3.0 %) as severely obese (≥30 kg/m(2)). As BMI increased, the relative hazard ratio (HR) decreased sharply, reaching the lowest value in the overweight group, and then rose again in the obese and severely obese. On univariate analysis, both overall survival (OS) and progression-free survival (PFS) were best in the overweight group, followed by normal > obese > severely obese > underweight groups. Multivariate analysis showed a significantly shorter survival in the underweight (OS: HR 2.90, 95 % confidence interval (CI) 1.35-6.19, P = 0.006; PFS: HR 3.08, 95 % CI 1.55-6.09, P = 0.001) and severely obese groups (OS: HR 2.93, 95 % CI 1.08-7.95, P = 0.035; PFS: HR 2.59, 95 % CI 1.06-6.36, P = 0.038). We show that being underweight or, contrary to findings in Western patients, being severely obese has a deleterious prognostic impact in DLBCL in Koreans. Revising the BMI criterion that defines obesity according to the patient's ethnic differences could therefore better delineate DLBCL risk groups in Asian patients.

Survivin, a member of the inhibitor of apoptosis protein family, is overexpressed in a variety of human neoplasms. The prognostic significance of survivin expression in diffuse large B-cell lymphoma patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) is unclear. We used standard immunohistochemistry methods to quantify survivin expression in 463 patients with de novo diffuse large B-cell lymphoma who received the R-CHOP. Of the 463 patients, 269 (58%) had survivin overexpression with a cutoff of >25%, associated with an International Prognostic Index score of >2 (P=0.015), disease in ≥2 extranodal sites (P=0.011), and a high Ki-67 index (P<0.0001). Among patients with activated B cell-like disease, the overall survival rate of survivin-positive patients was significantly lower than that of survivin-negative patients (P=0.033); multivariate analysis confirmed that in these patients, survivin overexpression was an independent prognostic factor for survival. Among patients with wild-type p53 overexpression, the overall survival and progression-free survival rates of the survivin-positive group were significantly lower than those of the survivin-negative group (P=0.035 and P=0.04 respectively). In STAT3-positive patients, survivin overexpression was associated with significantly better survival. Among patients with activated B cell-like disease, survivin-positive compared with survivin-negative groups had significantly different gene expression signatures, including genes involved in mitosis or tumor cell proliferation. Our results indicate that survivin is an independent prognostic factor for poor outcome in patients with activated B cell-like disease treated with the R-CHOP regimen, and patients with survivin-positive activated B cell-like diffuse large B-cell lymphoma seem to benefit less from this treatment and may require additional novel agents.

Dysregulated NF-κB signaling is critical for lymphomagenesis. The regulation, function, and clinical relevance of c-Rel/NF-κB activation in diffuse large B-cell lymphoma (DLBCL) have not been well studied. In this study we analyzed the prognostic significance and gene-expression signature of c-Rel nuclear expression as surrogate of c-Rel activation in 460 patients with de novo DLBCL. Nuclear c-Rel expression, observed in 137 (26.3%) DLBCL patients frequently associated with extranoal origin, did not show significantly prognostic impact in the overall- or germinal center B-like-DLBCL cohort, likely due to decreased pAKT and Myc levels, up-regulation of FOXP3, FOXO3, MEG3 and other tumor suppressors coincided with c-Rel nuclear expression, as well as the complicated relationships between NF-κB members and their overlapping function. However, c-Rel nuclear expression correlated with significantly poorer survival in p63+ and BCL-2- activated B-cell-like-DLBCL, and in DLBCL patients with TP53 mutations. Multivariate analysis indicated that after adjusting clinical parameters, c-Rel positivity was a significantly adverse prognostic factor in DLBCL patients with wild type TP53. Gene expression profiling suggested dysregulations of cell cycle, metabolism, adhesion, and migration associated with c-Rel activation. In contrast, REL amplification did not correlate with c-Rel nuclear expression and patient survival, likely due to co-amplification of genes that negatively regulate NF-κB activation. These insights into the expression, prognostic impact, regulation and function of c-Rel as well as its crosstalk with the p53 pathway underscore the importance of c-Rel and have significant therapeutic implications.

Nuclear factor-κB (NF-κB) is a transcription factor with a well-described oncogenic role. Study for each of five NF-κB pathway subunits was only reported on small cohorts in diffuse large B-cell lymphoma (DLBCL). In this large cohort (n=533) of patients with de novo DLBCL, we evaluated the protein expression frequency, gene expression signature, and clinical implication for each of these five NF-κB subunits. Expression of p50, p52, p65, RELB, and c-Rel was 34%, 12%, 20%, 14%, and 23%, whereas p50/p65, p50/c-Rel, and p52/RELB expression was 11%, 11%, and 3%, respectively. NF-κB subunits were expressed in both germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL, but p50 and p50/c-Rel were associated with ABC-DLBCL. p52, RELB, and p52/RELB expressions were associated with CD30 expression. p52 expression was negatively associated with BCL2 (B-cell lymphoma 2) expression and BCL2 rearrangement. Although p52 expression was associated with better progression-free survival (PFS) (P=0.0170), singular expression of the remaining NF-κB subunits alone did not show significant prognostic impact in the overall DLBCL cohort. Expression of p52/RELB was associated with better overall survival (OS) and PFS (P=0.0307 and P=0.0247). When cases were stratified into GCB- and ABC-DLBCL, p52 or p52/RELB dimer expression status was associated with better OS and PFS (P=0.0134 and P=0.0124) only within the GCB subtype. However, multivariate analysis did not show p52 expression to be an independent prognostic factor. Beneficial effect of p52 in GCB-DLBC appears to be its positive correlation with CD30 and negative correlation with BCL2 expression. Gene expression profiling (GEP) showed that p52(+) GCB-DLBCL was distinct from p52(-) GCB-DLBCL. Collectively, our data suggest that DLBCL patients with p52 expression might not benefit from therapy targeting the NF-κB pathway.

Epstein-Barr virus-positive diffuse large B-cell lymphoma of the elderly (EBV+ DLBCL-e) is a molecularly distinct variant of DLBCL, characterized by a monoclonal B-cell proliferation that occurs in patients >50 years of age without a history or clinicopathologic evidence of immunodeficiency. However, patients with EBV+ DLBCL younger than 50-years-old also exist in Western countries. We evaluated the clinicopathologic, immunophenotypic and genetic features in Cacausian patients with EBV+ DLBCL who are ≤50 years of age and compared this patient group to patients who are >50 years. In patients who are ≤50 years, less frequent expression of BCL6 and a trend of more frequent expression of CD30 and pSTAT3 were found in patients with EBV+ DLBCL. In patients who are >50 years, common expression of CD30, p50, pSTAT3 and less frequent expression of BCL6 were observed. Older patients also more commonly had a poor performance status (ECOG≥2). Comparing EBV+ DLBCL patients in ≤50 years versus >50 years, both groups had similar clinicopathologic, immunophenotypic and genetic features. Gene expression profiling, microRNA profiling and treatment outcome of the younger patients with EBV+ DLBCL was not distinctive from tumors in older patients. Based on our data, we suggest that the arbitrary age cutoff for EBV+ DLBCL is unnecessary and should be eliminated in the WHO lymphoma classification scheme.

Abnormal expression of the chemokine receptor CXCR4 plays an essential role in tumor cell dissemination and disease progression. However, the significance of CXCR4 overexpression in de novo diffuse large B cell lymphoma (DLBCL) is unknown. In 743 patients with de novo diffuse large B cell lymphoma (DLBCL) who received standard Rituximab-CHOP immunochemotherapy, we assessed the expression of CXCR4 and dissected its prognostic significance in various DLBCL subsets. Our results showed that CXCR4+ patients was associated with male, bulky tumor, high Ki-67 index, activated B-cell-like (ABC) subtype, and Myc, Bcl-2 or p53 overexpression. Moreover, CXCR4+ was an independent factor predicting poorer progression-free survival in germinal-center B-cell-like (GCB)-DLBCL, but not in ABC-DLBCL; and in patients with an IPI of ≤2, but not in those with an IPI>2. The lack of prognostic significance of CXCR4 in ABC-DLBCL was likely due to the activation of p53 tumor suppressor attenuating CXCR4 signaling. Furthermore, concurrent CXCR4+ and BCL2 translocation showed dismal outcomes resembling but independent of MYC/BCL2 double-hit DLBCL. Gene expression profiling suggested that alterations in the tumor microenvironment and immune responses, increased tumor proliferation and survival, and the dissemination of CXCR4+ tumor cells to distant organs or tissues were underlying molecular mechanisms responsible for the CXCR4+ associated poor prognosis.

CD5 is a pan-T-cell surface marker and is rarely expressed in diffuse large B-cell lymphoma (DLBCL). Large-scale studies of de novo CD5+ DLBCL are lacking in Western countries. In this study by the DLBCL Rituximab-CHOP Consortium, CD5 was expressed in 5.5% of 879 DLBCL patients from Western countries. CD5+ DLBCL was associated with higher frequencies of >1 ECOG performance status, bone marrow involvement, central nervous system relapse, activated B-cell-like subtype, Bcl-2 overexpression, and STAT3 and NF-κB activation, whereas rarely expressed single-stranded DNA-binding protein 2 (SSBP2), CD30 or had MYC mutations. With standard R-CHOP chemotherapy, CD5+ DLBCL patients had significantly worse overall survival (median, 25.3 months vs. not reached, P< .0001) and progression-free survival (median, 21.3 vs. 85.8 months, P< .0001) than CD5- DLBCL patients, which was independent of Bcl-2, STAT3, NF-κB and the International Prognostic Index. Interestingly, SSBP2 expression abolished the prognostic significance of CD5 expression, suggesting a tumor-suppressor role of SSBP2 for CD5 signaling. Gene-expression profiling demonstrated that B-cell receptor signaling dysfunction and microenvironment alterations are the important mechanisms underlying the clinical impact of CD5 expression. This study shows the distinctive clinical and biological features of CD5+ DLBCL patients in Western countries and underscores important pathways with therapeutic implications.

Background: Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) of the elderly is defined only in adults older than 50 years. However, EBV-positive DLBCL can affect younger patients. We investigated the prevalence, clinical characteristics and survival outcomes of EBV-positive DLBCL in young adults., Patients and Methods: We analyzed patients with de novo DLBCL who were registered in the Samsung Medical Center (SMC) retrospective lymphoma cohort and prospective SMC Lymphoma Cohort Study I (ClinicalTrials.gov: NCT00822731)., Results: A total of 571 cases were included in the analysis. The prevalence of EBV positivity was 6.7% (13/195) and 9.3% (35/376) in the young group (≤50 years) and in the elderly group (>50 years), respectively. EBV status was closely associated with unique unfavorable clinical characteristics [older age, more advanced stage, two or more sites of extranodal involvement, higher International Prognostic Index (IPI), and age-adjusted IPI risk] only in the elderly group. Poor prognostic impact of EBV positivity on overall survival was observed only in the elderly group [hazard ratio (HR) 2.86; 95% confidence interval (CI) 1.83-4.47; P < 0.001], but not in the young group (HR 1.17; 95% CI 0.35-3.89; P = 0.801)., Conclusion: A substantial proportion of EBV-positive DLBCL of the elderly can occur in young adults. EBV positivity of DLBCL in young adults was not associated with unfavorable clinical characteristics or worse outcomes. We suggest that EBV-positive DLBCL should not be confined only in the elderly and 'EBV-positive DLBCL in young adults' needs to be considered as a clinically distinct disease entity. ClinicalTrials.gov: NCT02060435., (© The Author 2014. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This study evaluated the effect of age and cell of origin on the prognostic significance of Ki-67 labeling index (Ki-67 LI) on overall survival (OS) of diffuse large B-cell lymphoma (DLBCL) in a cohort of 697 patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). Multivariate analysis revealed no prognostic significance of high Ki-67 LI (≥ 85%) for OS. However, on subgroup analysis, high Ki-67 LI was significantly associated with poor OS in late-elderly patients (aged ≥ 70 years) (p = 0.021) and non-germinal center B-cell-like (GCB) subtype (p = 0.015). In particular, high Ki-67 LI was associated with a poor prognosis in late-elderly patients with non-GCB subtype. No correlation was observed in young adults (aged < 60 years) or early-elderly (aged 60-70 years) patients or GCB subtype. The present study shows that high Ki-67 LI is a risk factor for poor OS in the late-elderly age group and non-GCB subtype in patients with DLBCL treated with R-CHOP.

Purpose: Activated signal transducer and activator of transcription 3 (STAT3) regulates tumor growth, invasion, cell proliferation, angiogenesis, immune response, and survival. Data regarding expression of phosphorylated (activated) STAT3 in diffuse large B-cell lymphoma (DLBCL) and the impact of phosphorylated STAT3 (pSTAT3) on prognosis are limited., Experimental Design: We evaluated expression of pSTAT3 in de novo DLBCL using immunohistochemistry, gene expression profiling (GEP), and gene set enrichment analysis (GSEA). Results were analyzed in correlation with cell-of-origin (COO), critical lymphoma biomarkers, and genetic translocations., Results: pSTAT3 expression was observed in 16% of DLBCL and was associated with advanced stage, multiple extranodal sites of involvement, activated B-cell-like (ABC) subtype, MYC expression, and MYC/BCL2 expression. Expression of pSTAT3 predicted inferior overall survival (OS) and progression-free survival (PFS) in patients with de novo DLBCL. When DLBCL cases were stratified according to COO or MYC expression, pSTAT3 expression did not predict inferior outcome, respectively. Multivariate analysis showed that the prognostic predictability of pSTAT3 expression was due to its association with the ABC subtype, MYC expression, and adverse clinical features. GEP demonstrated upregulation of genes, which can potentiate function of STAT3. GSEA showed the JAK-STAT pathway to be enriched in pSTAT3(+) DLBCL., Conclusions: The results of this study provide a rationale for the ongoing successful clinical trials targeting the JAK-STAT pathway in DLBCL., (©2014 American Association for Cancer Research.)

Objectives: A recent study suggested a prognostic role for the peripheral blood absolute lymphocyte/monocyte ratio (LMR) at diagnosis of diffuse large B-cell lymphoma (DLBCL). Here, we investigated the significance of LMR in DLBCL patients in relation to advanced age., Methods: We examined the prognostic impact of LMR in 603 DLBCL treated with rituximab plus CHOP, using the receiver operating characteristic curve analysis for optimal cut-off values, and performed a subgroup analysis according to age., Results: In elderly groups (age ≥ 70), absolute monocyte count was significantly increased, whereas LMR was significantly decreased compared to younger groups. Patients under 70 yr of age with LMR < 3.04 had significantly lower overall survival (OS) and progression-free survival (PFS) compared to those with LMR ≥ 3.04 (P < 0.001 for both). However, in elderly patients (age ≥ 70), there was no significant difference in OS between patients' LMR levels using the 3.04 cut-off value (P = 0.059). Therefore, a new LMR cut-off value of 2.36 was selected in elderly patients, having observed that elderly patients with LMR < 2.36 had significantly lower OS compared to those with LMR ≥ 2.36 (P = 0.021). In multivariate analysis, LMR remained a significant prognostic factor for OS (P = 0.004) or PFS (P < 0.001)., Conclusions: We suggest the use of a different cut-off value of LMR in elderly patients to distinguish high-risk from low-risk groups., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Although the International Prognostic Index (IPI) is considered as the current standard prognostication system for diffuse large B-cell lymphoma (DLBCL), prognostic heterogeneity is suggested to exist among the patients within the same IPI risk group. Hence, we investigated the pattern of distribution and prognostic impact of five IPI factors within the same IPI score. We retrospectively reviewed the medical records of 387 patients newly diagnosed as pathologically proven DLBCL between February 2002 and February 2010. We classified patients to IPI risk scores and categorized them according to the combinations of IPI. Then, we explored the frequency of five IPI factors and analyzed the correlation between these subgroups and efficacy outcomes: complete response (CR), event-free survival (EFS), and overall survival (OS). Survival estimates by IPI score in this cohort corresponded to the classic IPI. Elevated serum level of lactate dehydrogenase (LDH) was the most prevalently distributed factor throughout the scores, and patients with elevated serum level of LDH tended to have lower CR, inferior EFS, and/or OS irrespective of IPI scores. Particularly, among the subgroups of IPI score of 2, elevated serum level of LDH was significantly associated with inferior CR (73.1 vs 95.2 %), 3-year EFS (57 vs 87 %), and 3-year OS (58 vs 82 %). In addition, the higher serum level of LDH, particularly above 2,000 IU/L, was significantly correlated with the inferior survival outcomes (3-year EFS 78.0 vs 58.5 vs 45.5 vs 20.0 %, 3-year OS 86.0 vs 66.2 vs 58.2 vs 40.0 %). In conclusion, among five factors of IPI, elevated serum level of LDH seems to be the most frequently distributed and, more importantly, the most relevant IPI factor with the highest prognostic impact. These findings still warrant further validation in larger cohorts.

Diffuse large B-cell lymphoma (DLBCL) is classified into prognostically distinct germinal center B-cell (GCB) and activated B-cell subtypes by gene expression profiling (GEP). Recent reports suggest the role of GEP subtypes in targeted therapy. Immunohistochemistry (IHC) algorithms have been proposed as surrogates of GEP, but their utility remains controversial. Using microarray, we examined the concordance of 4 GEP-correlated and 2 non-GEP-correlated IHC algorithms in 381 DLBCLs, not otherwise specified. Subtypes and variants of DLBCL were excluded to minimize the possible confounding effect on prognosis and phenotype. Survival was analyzed in 138 cyclophosphamide, adriamycin, vincristine, and prednisone (CHOP)-treated and 147 rituximab plus CHOP (R-CHOP)-treated patients. Of the GEP-correlated algorithms, high concordance was observed among Hans, Choi, and Visco-Young algorithms (total concordance, 87.1%; κ score: 0.726 to 0.889), whereas Tally algorithm exhibited slightly lower concordance (total concordance 77.4%; κ score: 0.502 to 0.643). Two non-GEP-correlated algorithms (Muris and Nyman) exhibited poor concordance. Compared with the Western data, incidence of the non-GCB subtype was higher in all algorithms. Univariate analysis showed prognostic significance for Hans, Choi, and Visco-Young algorithms and BCL6, GCET1, LMO2, and BCL2 in CHOP-treated patients. On multivariate analysis, Hans algorithm retained its prognostic significance. By contrast, neither the algorithms nor individual antigens predicted survival in R-CHOP treatment. The high concordance among GEP-correlated algorithms suggests their usefulness as reliable discriminators of molecular subtype in DLBCL, not otherwise specified. Our study also indicates that prognostic significance of IHC algorithms may be limited in R-CHOP-treated Asian patients because of the predominance of the non-GCB type.