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A significant challenge in the core modeling of pebble bed reactors (PBRs) is the complex fuel-coolant structure. At the expense of approximating local flow and heat transfer effects, porous media models can provide medium-fidelity predictions of complicated thermal-fluid systems with significantly less computational cost than high-fidelity Computational Fluid Dynamics (CFD) models. This paper presents a new porous media code, Pronghorn – a fast-running core simulator intended to accelerate the design and analysis cycle for PBRs and provide boundary conditions for systems-level analysis. This paper describes the physical models in Pronghorn and demonstrates the capability of a friction-dominated model for predicting gas-cooled PBR decay heat removal by presenting simulation results for all 52 of the steady-state axisymmetric German SANA experiments, which include two different fluids and three different types of pebbles. The pebble temperature in all 52 cases is predicted with a mean error (predicted minus experimental) of +22.6 °C with standard deviation of 54.6 °C. To demonstrate Pronghorn's capability for modeling bed-to-plenum heat and mass transfer, one open-plenum SANA experimental case is also simulated. A code-to-code comparison with Flownex and GAMMA shows that Pronghorn is comparable in accuracy to other porous media simulation tools, with the additional advantages of 1) an arbitrary equation of state; 2) 3-D unstructured mesh capabilities; and 3) multiphysics coupling to other Multiphysics Object-Oriented Simulation Environment (MOOSE) applications. Finally, the effect of several porous media closure selections, in particular the porosity, the near-wall treatment for effective solid thermal conductivity, the interphase drag and heat transfer, and the fluid thermal dispersion, on temperature predictions are quantified.

Pebble bed High Temperature Reactors (HTRs) are characterized by many advantageous design features, such as excellent passive heat removal in accidents, large margins to fuel failure, and online refueling potential. However, a significant challenge in the core modeling of pebble bed reactors is the complex fuel-coolant structure. This paper presents a new porous media simulation code, Pronghorn, that aims to alleviate modeling challenges for pebble bed reactors by providing a fast-running, mediumfidelity core simulator. Pronghorn is intended to accelerate the design and analysis cycle for pebble bed and prismatic HTRs by permitting fast scoping studies and providing boundary conditions for systems-level analysis. Pronghorn is built on the Multiphysics Object-Oriented Simulation Environment (MOOSE) using modern software practices and a thorough testing framework. This paper describes the physical models used in Pronghorn and demonstrates Pronghorn's capability for modeling gas-cooled pebble bed HTRs by presenting simulation results obtained for the German SANA pebble bed decay heat experiments. Within the limitations of the porous media approximation and existing available closure relationships, Pronghorn predicts the SANA experimental pebble temperatures well, expanding the code's validation base. A brief code-To-code comparison shows a level of accuracy comparable to other porous media simulation tools. Pronghorn's advantages over these related tools include: An arbitrary equation of state, unstructured mesh capabilities, compressible flow models, the ability to couple to MOOSE fuels performance and systems-level thermal-hydraulics codes, and modern software design.

Pebble bed High Temperature Reactors (HTRs) are characterized by many advantageous design features, such as excellent passive heat removal in accidents and large margins to fuel failure. However, a significant challenge in thermal-hydraulic core modeling of pebble bed reactors is the double heterogeneity random packing of hundreds of thousands of fuel pebbles and thousands of fuel particles per pebble. A new porous media thermal-hydraulics code, Pronghorn, is under development to provide a fast-running, medium-fidelity core simulator and serve as a bridge between low-resolution system level codes and high-resolution Computational Fluid Dynamics (CFD) codes for multiscale analysis. Pronghorn is based on the Mul-tiphysics Object-Oriented Simulation Environment (MOOSE) finite element framework, and permits an arbitrary equation of state, unstructured mesh capabilities, modern software design, and the ability to couple to MOOSE fuels performance and systems-level Thermal-Hydraulic (T/H) codes. To address the wide variety in gas- and liquid-cooled pebble bed reactor designs, Pronghorn includes several different flow models, each most appropriate to a range of compressibilities and operating conditions. This paper reviews benchmarking efforts of a low-advection flow model appropriate for Loss of Forced Circulation (LOFC) simulations and introduces the new fully compressible flow model with preliminary validation by comparison to potential flow theory for low Mach number flow over a cylinder.

This study sheds light on the pivotal role of the oncoprotein DEK in B-cell lymphoma. We reveal DEK expression correlates with increased tumor proliferation and inferior overall survival in cases diagnosed with low-grade B-cell lymphoma (LGBCL). We also found significant correlation between DEK expression and copy number alterations in LGBCL tumors, highlighting a novel mechanism of LGBCL pathogenesis that warrants additional exploration. To interrogate the mechanistic role of DEK in B-cell lymphoma, we generated a DEK knockout cell line model, which demonstrated DEK depletion caused reduced proliferation and altered expression of key cell cycle and apoptosis-related proteins, including Bcl-2, Bcl-xL, and p53. Notably, DEK depleted cells showed increased sensitivity to apoptosis-inducing agents, including venetoclax and staurosporine, which underscores the therapeutic potential of targeting DEK in B-cell lymphomas. Overall, our study contributes to a better understanding of DEK's role as an oncoprotein in B-cell lymphomas, highlighting its potential as both a promising therapeutic target and a novel biomarker for aggressive LGBCL. Further research elucidating the molecular mechanisms underlying DEK-mediated tumorigenesis could pave the way for improved treatment strategies and better clinical outcomes for patients with B-cell lymphoma., (© 2024. The Author(s).)

Abstract: Currently, the role of DNA methylation in the immunoglobulin M (IgM) monoclonal gammopathy disease spectrum remains poorly understood. In the present study, a multiomics prospective analysis was conducted integrating DNA methylation, RNA sequencing (RNA-seq), and whole-exome sequencing data in 34 subjects (23 with Waldenström macroglobulinemia [WM], 6 with IgM monoclonal gammopathy of undetermined significance [MGUS], and 5 normal controls). Analysis was focused on defining differences between IgM gammopathies (WM/IgM-MGUS) compared with controls, and specifically between WM and IgM-MGUS. Between groups, genome-wide DNA methylation analysis demonstrated a significant number of differentially methylated regions that were annotated according to genomic region. Next, integration of RNA-seq data was performed to identify potentially epigenetically deregulated pathways. We found that pathways involved in cell cycle, metabolism, cytokine/immune signaling, cytoskeleton, tumor microenvironment, and intracellular signaling were differentially activated and potentially epigenetically regulated. Importantly, there was a positive enrichment of the CXCR4 signaling pathway along with several interleukin (interleukin 6 [IL-6], IL-8, and IL-15) signaling pathways in WM compared with IgM-MGUS. Further assessment of known tumor suppressor genes and oncogenes uncovered differential promoter methylation of several targets with concordant change in gene expression, including CCND1 and CD79B. Overall, this report defines how aberrant DNA methylation in IgM gammopathies may play a critical role in the epigenetic control of oncogenesis and key cellular functions., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Immunochemotherapy has been the mainstay of treatment for newly diagnosed diffuse large B-cell lymphoma (ndDLBCL) yet is inadequate for many patients. In this work, we perform unsupervised clustering on transcriptomic features from a large cohort of ndDLBCL patients and identify seven clusters, one called A7 with poor prognosis, and develop a classifier to identify these clusters in independent ndDLBCL cohorts. This high-risk cluster is enriched for activated B-cell cell-of-origin, low immune infiltration, high MYC expression, and copy number aberrations. We compare and contrast our methodology with recent DLBCL classifiers to contextualize our clusters and show improved prognostic utility. Finally, using pre-clinical models, we demonstrate a mechanistic rationale for IKZF1/3 degraders such as lenalidomide to overcome the low immune infiltration phenotype of A7 by inducing T-cell trafficking into tumors and upregulating MHC I and II on tumor cells, and demonstrate that TCF4 is an important regulator of MYC-related biology in A7., (© 2024. The Author(s).)

Recent genetic and molecular classification of DLBCL has advanced our knowledge of disease biology, yet were not designed to predict early events and guide anticipatory selection of novel therapies. To address this unmet need, we used an integrative multiomic approach to identify a signature at diagnosis that will identify DLBCL at high risk of early clinical failure. Tumor biopsies from 444 newly diagnosed DLBCL were analyzed by WES and RNAseq. A combination of weighted gene correlation network analysis and differential gene expression analysis was used to identify a signature associated with high risk of early clinical failure independent of IPI and COO. Further analysis revealed the signature was associated with metabolic reprogramming and identified cases with a depleted immune microenvironment. Finally, WES data was integrated into the signature and we found that inclusion of ARID1A mutations resulted in identification of 45% of cases with an early clinical failure which was validated in external DLBCL cohorts. This novel and integrative approach is the first to identify a signature at diagnosis, in a real-world cohort of DLBCL, that identifies patients at high risk for early clinical failure and may have significant implications for design of therapeutic options., (© 2024. The Author(s).)

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8 + CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8 + T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8 + CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies., (© 2024. The Author(s).)

Follicular lymphoma (FL) is an indolent non-Hodgkin lymphoma of germinal center origin, which presents with significant biologic and clinical heterogeneity. Using RNA-seq on B cells sorted from 87 FL biopsies, combined with machine-learning approaches, we identify 3 transcriptional states that divide the biological ontology of FL B cells into inflamed, proliferative, and chromatin-modifying states, with relationship to prior GC B cell phenotypes. When integrated with whole-exome sequencing and immune profiling, we find that each state was associated with a combination of mutations in chromatin modifiers, copy-number alterations to TNFAIP3, and T follicular helper cells (Tfh) cell interactions, or primarily by a microenvironment rich in activated T cells. Altogether, these data define FL B cell transcriptional states across a large cohort of patients, contribute to our understanding of FL heterogeneity at the tumor cell level, and provide a foundation for guiding therapeutic intervention., Competing Interests: Declaration of interests A.J.N. has received research funding from Bristol Myers Squibb., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Non-follicular low-grade B-cell lymphomas (LGBCL) are biologically diverse entities that share clinical and histologic features that make definitive pathologic categorization challenging. While most patients with LGBCL have an indolent course, some experience aggressive disease, highlighting additional heterogeneity across these subtypes. To investigate the potential for shared biology across subtypes, we performed RNA sequencing and applied machine learning approaches that identified five clusters of patients that grouped independently of subtype. One cluster was characterized by inferior outcome, upregulation of cell cycle genes, and increased tumor immune cell content. Integration of whole exome sequencing identified novel LGBCL mutations and enrichment of TNFAIP3 and BCL2 alterations in the poor survival cluster. Building on this, we further refined a transcriptomic signature associated with early clinical failure in two independent cohorts. Taken together, this study identifies unique clusters of LGBCL defined by novel gene expression signatures and immune profiles associated with outcome across diagnostic subtypes., (© 2023 The Authors. Hematological Oncology published by John Wiley & Sons Ltd.)

T-lymphocytes are prevalent in the tumor microenvironment of follicular lymphoma (FL). However, the phenotype of T-cells may vary, and the prevalence of certain T-cell subsets may influence tumor biology and patient survival. We therefore analyzed a cohort of 82 FL patients using CyTOF to determine whether specific T-cell phenotypes were associated with distinct tumor microenvironments and patient outcome. We identified four immune subgroups with differing T-cell phenotypes and the prevalence of certain T-cell subsets was associated with patient survival. Patients with increased T cells with early differentiation stage tended to have a significantly better survival than patients with increased T-cells of late differentiation stage. Specifically, CD57 + T FH cells, with a late-stage differentiation phenotype, were significantly more abundant in FL patients who had early disease progression and therefore correlated with an inferior survival. Single cell analysis (CITE-seq) revealed that CD57 + T FH cells exhibited a substantially different transcriptome from CD57 - T FH cells with upregulation of inflammatory pathways, evidence of immune exhaustion and susceptibility to apoptosis. Taken together, our results show that different tumor microenvironments among FL patients are associated with variable T-cell phenotypes and an increased prevalence of CD57 + T FH cells is associated with poor patient survival., (© 2023. Springer Nature Limited.)

The role of the tumor microenvironment (TME) and intratumoral T cells in splenic marginal zone lymphoma (sMZL) is largely unknown. In the present study, we evaluated 36 sMZL spleen specimens by single cell analysis to gain a better understanding of the TME in sMZL. Using mass cytometry (CyTOF), we observed that the TME in sMZL is distinct from that of control non-malignant reactive spleen (rSP). We found that the number of T FH cells varied greatly in sMZL, ICOS + T FH cells were more abundant in sMZL than rSP, and T FH cells positively correlated with increased numbers of memory B cells. T reg cell analysis revealed that TIGIT + T reg cells are enriched in sMZL and correlate with suppression of T H 17 and T H 22 cells. Intratumoral CD8 + T cells were comprised of subsets of short-lived, exhausted and late-stage differentiated cells, thereby functionally impaired. We observed that T-cell exhaustion was present in sMZL and TIM-3 expression on PD-1 low cells identified cells with severe immune dysfunction. Gene expression profiling by CITE-seq analysis validated this finding. Taken together, our data suggest that the TME as a whole, and T-cell population specifically, are heterogenous in sMZL and immune exhaustion is one of the major factors impairing T-cell function., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

How to identify follicular lymphoma (FL) patients with low disease burden but high risk for early progression is unclear. Building on a prior study demonstrating the early transformation of FLs with high variant allele frequency (VAF) BCL2 mutations at activation-induced cytidine deaminase (AICDA) sites, we examined 11 AICDA mutational targets, including BCL2, BCL6, PAX5, PIM1, RHOH, SOCS, and MYC, in 199 newly diagnosed grade 1 and 2 FLs. BCL2 mutations with VAF ≥20% occurred in 52% of cases. Among 97 FL patients who did not initially receive rituximab-containing therapy, nonsynonymous BCL2 mutations at VAF ≥20% were associated with increased transformation risk (HR 3.01, 95% CI 1.04-8.78, p = 0.043) and a trend toward shorter event-free survival (EFS, median 20 months with mutations versus 54 months without, p = 0.052). Other sequenced genes were less frequently mutated and did not increase the prognostic value of the panel. Across the entire population, nonsynonymous BCL2 mutations at VAF ≥20% were associated with decreased EFS (HR 1.55, 95% CI 1.02-2.35, p = 0.043 after correction for FLIPI and treatment) and decreased overall survival after median 14-year follow-up (HR 1.82, 95% CI 1.05-3.17, p = 0.034). Thus, high VAF nonsynonymous BCL2 mutations remain prognostic even in the chemoimmunotherapy era., (© 2023. The Author(s).)

Purpose: IgM monoclonal gammopathy of undetermined significance (MGUS) and Waldenström macroglobulinemia (WM) represent a disease spectrum with highly varied therapeutic management, ranging from observation to chemoimmunotherapy. The current classification relies solely on clinical features and does not explain the heterogeneity that exists within each of these conditions. Further investigation is warranted to shed light on the biology that may account for the clinical differences., Experimental Design: We used bone marrow (BM) clonal CD19+ and/or CD138+ sorted cells, matched BM supernatant, and peripheral blood serum from 32 patients (7 MGUS, 25 WM) to perform the first multi-omics approach including whole-exome sequencing, RNA sequencing, proteomics, metabolomics, and mass cytometry., Results: We identified three clusters with distinct pathway activation, immune content, metabolomic, and clinical features. Cluster 1 included only patients with WM and was characterized by transcriptional silencing of genes involved in cell cycle and immune response, enrichment of mitochondrial metabolism, infiltration of senescent T effector memory cells, and aggressive clinical behavior. Genetic/structural alterations of TNFAIP3 were distinct events of this cluster. Cluster 2 comprised both MGUS and WM patients with upregulation of inflammatory response, senescence and glycolysis signatures, increased activated T follicular helper and T regulatory cells, and indolent clinical behavior. Cluster 3 also included both MGUS and WM patients and exhibited intermediate features, including proliferative and inflammatory signaling, as well as glycolysis and mitochondrial metabolism., Conclusions: We have identified three distinct molecular clusters, suggesting a potential biologic classification that may have therapeutic implications., (©2022 American Association for Cancer Research.)

Lymphoma risk is elevated for relatives with common non-Hodgkin lymphoma (NHL) subtypes, suggesting shared genetic susceptibility across subtypes. To evaluate the extent of mutual heritability among NHL subtypes and discover novel loci shared among subtypes, we analyzed data from eight genome-wide association studies within the InterLymph Consortium, including 10,629 cases and 9505 controls. We utilized Association analysis based on SubSETs (ASSET) to discover loci for subsets of NHL subtypes and evaluated shared heritability across the genome using Genome-wide Complex Trait Analysis (GCTA) and polygenic risk scores. We discovered 17 genome-wide significant loci (P < 5 × 10 -8 ) for subsets of NHL subtypes, including a novel locus at 10q23.33 (HHEX) (P = 3.27 × 10 -9 ). Most subset associations were driven primarily by only one subtype. Genome-wide genetic correlations between pairs of subtypes varied broadly from 0.20 to 0.86, suggesting substantial heterogeneity in the extent of shared heritability among subtypes. Polygenic risk score analyses of established loci for different lymphoid malignancies identified strong associations with some NHL subtypes (P < 5 × 10 -8 ), but weak or null associations with others. Although our analyses suggest partially shared heritability and biological pathways, they reveal substantial heterogeneity among NHL subtypes with each having its own distinct germline genetic architecture., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Purpose: Regulatory T-cells (Treg) are essential to Tregs homeostasis and modulate the antitumor immune response in patients with lymphoma. However, the biology and prognostic impact of Tregs in splenic marginal zone lymphoma (SMZL) have not been studied., Experimental Design: Biopsy specimens from 24 patients with SMZL and 12 reactive spleens (rSP) from individuals without lymphoma were analyzed by using CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), CyTOF (mass cytometry) analysis, and flow cytometry to explore the phenotype, transcriptomic profile, and clinical significance of intratumoral Tregs and their subsets. The biological characteristics and cell signaling pathways of intratumoral Treg subsets were confirmed by in vitro functional assays., Results: We found that Tregs are more abundant in SMZL patients' spleens than rSP, and Tregs from patients with SMZL and rSP can be separated into CD161+Treg and CD26+Treg subsets. CD161+Tregs are increased in SMZL but have dysregulated immune function. We found that CD161+Treg and CD26+Tregs have unique gene expression and phenotypic profiles and are differentially correlated with patient outcomes. Specifically, increased CD161+Tregs are significantly associated with a favorable prognosis in patients with SMZL, whereas CD26+Tregs are associated with a poor prognosis. Furthermore, activation of the IL2/STAT5 pathway contributes to the induction of CD26+Tregs and can be reversed by STAT5 inhibition., Conclusions: IL2/STAT5-mediated expansion of CD26+Tregs contributes to a poor clinical outcome in SMZL and may represent a therapeutic opportunity in this disease., (©2022 American Association for Cancer Research.)

Low-grade B-cell lymphomas other than follicular and small lymphocytic lymphoma (LGBCL) account for 10% of all B-cell non-Hodgkin lymphomas. Despite improvements in survival outcomes for these patients, little is known about cause of death (COD) in the rituximab era. For a better understanding, we studied 822 newly diagnosed patients with marginal zone, lymphoplasmacytic, and unclassifiable low-grade B-cell lymphoma prospectively enrolled in the University of Iowa/Mayo Clinic Specialized Program of Research Excellence Molecular Epidemiology Resource from 2002 to 2015. COD was assigned based on medical record review using a standard protocol. At a median follow-up of 107 months, 219 (27%) patients had died. The incidence of lymphoma-related deaths when pooling across subtypes was lower than non-lymphoma-related deaths (10-year incidence, 8.0%; 95% confidence interval [CI]: 6.2-10.4 vs 13.6%; 95% CI: 11.2-16.6). The incidence of lymphoma-related deaths varied by subtype, ranging from 3.7% at 10 years in extranodal marginal zone lymphoma to 19.3% in lymphoplasmacytic lymphoma/Waldenström macroglobulinemia. Patients with early progression or retreatment events, defined using event-free survival at 24 months from diagnosis, had significantly higher likelihood of lymphoma-related death compared with patients without early events (10-year estimate: 19.1% vs 5.1%, respectively; P < .001), whereas the rates for non-lymphoma-related death were comparable in patients with or without early events (10-year estimates: 11.0% vs 15.3%, respectively). In conclusion, the most common COD in LGBCLs in the first decade after diagnosis was for causes other than lymphoma. Progression or retreatment within the first 2 years of diagnosis was a strong predictor for risk of lymphoma-related death., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Vaccinations have been hypothesized to play a role in lymphoma etiology, but there are few studies, mixed results, and limited data on lymphoma subtypes. Herein, we investigate the association of vaccinations with risk of major lymphoma subtypes., Methods: We studied 2,461 lymphoma cases and 2,253 controls enrolled from 2002 to 2014. Participants self-reported history of vaccinations against hepatitis A, hepatitis B, yellow fever, and influenza. Polytomous logistic regression was used to estimate OR and 95% confidence intervals (CI), adjusting for potential confounders., Results: After multivariable adjustment, vaccination against influenza was inversely associated with lymphoma (OR = 0.82; 95% CI, 0.66-1.02), which was stronger for last vaccination 1+ years before enrollment (OR = 0.71; 95% CI, 0.56-0.91) and for >5 influenza vaccinations (OR = 0.56; 95% CI, 0.46-0.68). Ever vaccination against hepatitis A (OR = 0.81; 95% CI, 0.66-1.00) but not hepatitis B (OR = 0.97; 95% CI, 0.81-1.18) was associated with lymphoma risk, although more recent vaccinations were inversely associated with lymphoma risk for both hepatitis A (<6 years before enrollment, OR = 0.56; 95% CI, 0.40-0.77) and hepatitis B (<9 years before enrollment, OR = 0.72; 95% CI, 0.55-0.93). Ever vaccination against yellow fever was inversely associated with risk (OR = 0.73; 95% CI, 0.55-0.96), and this did not vary by time since last vaccination. Although there was no overall statistical evidence for heterogeneity of vaccination history by lymphoma subtype, the only statistically significant inverse associations were observed for influenza and yellow fever vaccinations with diffuse large B-cell and follicular lymphoma., Conclusions: Selected vaccinations were inversely associated with lymphoma risk, with time since last vaccination relevant for some of these vaccines., Impact: Vaccinations against hepatitis A, hepatitis B, yellow fever, and influenza are unlikely to increase lymphoma risk., (©2021 American Association for Cancer Research.)

Despite a characteristic indolent course, a substantial subset of follicular lymphoma (FL) patients has an early relapse with a poor outcome. Cells in the microenvironment may be a key contributor to treatment failure. We used a discovery and validation study design to identify microenvironmental determinants of early failure and then integrated these results into the FLIPI. In total, 496 newly diagnosed FL grade 1-3 A patients who were prospectively enrolled into the MER cohort from 2002 to 2012 were evaluated. Tissue microarrays were stained for CD4, CD8, FOXP3, CD32b, CD14, CD68, CD70, SIRP-α, TIM3, PD-1, and PD-L1. Early failure was defined as failing to achieve event-free survival at 24 months (EFS24) in immunochemotherapy-treated patients and EFS12 in all others. CyTOF and CODEX analysis were performed to characterize intratumoral immunophenotypes. Lack of intrafollicular CD4 expression was the only predictor of early failure that replicated with a pooled OR 2.37 (95%CI 1.48-3.79). We next developed a bio-clinical risk model (BioFLIPI), where lack of CD4 intrafollicular expression moved patients up one FLIPI risk group, adding a new fourth high-risk group. Compared with BioFLIPI score of 1, patients with a score of 2 (OR 2.17; 95% CI 1.08-4.69), 3 (OR 3.53; 95% CI 1.78-7.54), and 4 (OR 8.92; 95% CI 4.00-21.1) had increasing risk of early failure. The favorable intrafollicular CD4 T cells were identified as activated central memory T cells, whose prognostic value was independent from genetic features. In conclusion, lack of intrafollicular CD4 expression predicts early failure in FL and combined with FLIPI improves identification of high-risk patients; however, independent validation is warranted., (© 2021. The Author(s).)

Background: CD8 + T-lymphocyte subsets defined by killer lectin-like receptor G1 (KLRG1) and CD127 expression have been reported to have an important role in infection, but their role in the setting of lymphoid malignancies, specifically follicular lymphoma (FL), has not been studied., Methods: To characterize the phenotype of KLRG1/CD127-defined CD8 + subsets, surface and intracellular markers were measured by flow cytometry and Cytometry by time of flight (CyTOF), and the transcriptional profile of these cells was determined by CITE-Seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing). The functional capacity of each subset was determined, as was their impact on overall survival (OS) and event-free survival (EFS) of patients with FL., Results: We found that intratumoral CD8 + cells in FL are skewed toward effector cell subsets, particularly KLRG + CD127 - and KLRG1 - CD127 - cells over memory cell subsets, such as KLRG1 - CD127 + and KLRG1 + CD127 + cells. While effector subsets exhibited increased capacity to produce cytokines/granules when compared with memory subsets, their proliferative capacity and viability were found to be substantially inferior. Clinically, a skewed distribution of intratumoral CD8 + T cells favoring effector subtypes was associated with an inferior outcome in patients with FL. Increased numbers of CD127 + KLRG1 - and CD127 + KLRG1 + were significantly associated with a favorable OS and EFS, while CD127 - KLRG1 - correlated with a poor EFS and OS in patients with FL. Furthermore, we demonstrated that interleukin (IL)-15 promotes CD127 - KLRG1 + cell development in the presence of dendritic cells via a phosphoinositide 3-kinase (PI3K)-dependent mechanism, and treatment of CD8 + T cells with a PI3K inhibitor downregulated the transcription factors responsible for CD127 - KLRG1 + differentiation., Conclusions: Taken together, these results reveal not only a biological and prognostic role for KLRG1/CD127-defined CD8 + subsets in FL but also a potential role for PI3K inhibitors to manipulate the differentiation of CD8 + T cells, thereby promoting a more effective antitumor immune response., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. 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 the most common lymphoma, and front line therapies have not improved overall outcomes since the advent of immunochemotherapy. By pairing DNA and gene expression data with clinical response data, we identified a high-risk subset of non-GCB DLBCL patients characterized by genomic alterations and expression signatures capable of sustaining an inflammatory environment. These mutational alterations (PIM1, SPEN, and MYD88 [L265P]) and expression signatures (NF-κB, IRF4, and JAK-STAT engagement) were associated with proliferative signaling, and were found to be enriched in patients treated with RCHOP that experienced unfavorable outcomes. However, patients with these high-risk mutations had more favorable outcomes when the immunomodulatory agent lenalidomide was added to RCHOP (R2CHOP). We are the first to report the genomic validation of a high-risk phenotype with a preferential response towards R2CHOP therapy in non-GCB DLBCL patients. These conclusions could be translated to a clinical setting to identify the ~38% of non-GCB patients that could be considered high-risk, and would benefit from alternative therapies to standard RCHOP based on personalized genomic data.

Waldenstrom macroglobulinemia (WM) has an annual incidence of 3-3.2 cases per million-person/year. National Cancer Data Base was used to identify newly diagnosed WM cases requiring initiation of therapy and their annual facility volume was used to divide the treatment facilities into four quartiles (Qs). Cox regression was used to analyze the association between facility volume and survival, adjusted by demographics, socioeconomic, geographic, comorbidity factors and year of diagnosis. A total of 3064 patients treated in 795 facilities were included. The unadjusted median overall survival (OS) by facility volume was: Q1:6.5 years (5-year OS 55%), Q2:7 years (5-year OS 60%), Q3:8 years (5-year OS 64%), and Q4: NR (5-year OS 71%), p  < 0.0001. Our results demonstrated that a volume-outcome relationship exists in WM and is an independent predictor of overall survival in addition to the established risk factors as age and disease severity.

TNFα is implicated in chronic lymphocytic leukemia (CLL) immunosuppression and disease progression. TNFα is constitutively produced by CLL B cells and is a negative regulator of bone marrow (BM) myelopoiesis. Here, we show that co-culture of CLL B cells with purified normal human hematopoietic stem and progenitor cells (HSPCs) directly altered protein levels of the myeloid and erythroid cell fate determinants PU.1 and GATA-2 at the single-cell level within transitional HSPC subsets, mimicking ex vivo expression patterns. Physical separation of CLL cells from control HSPCs or neutralizing TNFα abrogated upregulation of PU.1, yet restoration of GATA-2 required TNFα neutralization, suggesting both cell contact and soluble-factor-mediated regulation. We further show that CLL patient BM myeloid progenitors are diminished in frequency and function, an effect recapitulated by chronic exposure of control HSPCs to low-dose TNFα. These findings implicate CLL B-cell-derived TNFα in impaired BM myelopoiesis., Competing Interests: Research funding to S.A.P. has been provided from 10.13039/100014491Pharmacyclics, 10.13039/501100013650MorphoSys, Janssen, 10.13039/100004325AstraZeneca, 10.13039/100013252TG Therapeutics, 10.13039/100006436Celgene, 10.13039/100006483AbbVie, and Ascentage Pharma for clinical studies in which S.A.P. is a principal investigator. S.A.P. also participated in Advisory Board meetings of Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie (he was not personally compensated for his participation). Research funding to N.E.K. has been provided from Acerta Pharma BV, 10.13039/100006436Celgene, 10.13039/100004328Genentech, 10.13039/100014491Pharmacyclics, and Tolero Pharmaceutical. N.E.K. also participates in the data safety monitoring committees of Agios Pharm, Astra Zeneca, Celgene, Cytomx Therapeutics, Genentech, Infinity Pharm, Morpho-Sys, and Pharmacyclics (he was not personally compensated for his participation). All other authors declare no competing interests., (© 2020 The Author(s).)

Double/triple hit lymphoma (DH/TH), known as high-grade B-cell lymphoma (HGBL), is an aggressive diffuse large B cell lymphoma (DLBCL), defined as having concurrent MYC, BCL2, and/or BCL6 gene rearrangements. While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, and their contributions to rearrangements have yet to be fully elucidated. Using FISH and high-resolution CNA data, we defined the landscape of concurrent gene rearrangements and copy gains in MYC, BCL2, and BCL6, in a cohort of 479 newly diagnosed DLBCL. We also show that concurrent translocations and copy number alterations, in combinations similar to DH/TH, identify a unique subset of DLBCL, alternative DH/TH, that have survival outcomes similar to DH/TH DLBCL patients.

Purpose: TNF-related apoptosis inducing ligand (TRAIL) expression by immune cells contributes to antitumor immunity. A naturally occurring splice variant of TRAIL, called TRAILshort, antagonizes TRAIL-dependent cell killing. It is unknown whether tumor cells express TRAILshort and if it impacts antitumor immunity., Experimental Design: We used an unbiased informatics approach to identify TRAILshort expression in primary human cancers, and validated those results with IHC and ISH. TRAILshort-specific mAbs were used to determine the effect of TRAILshort on tumor cell sensitivity to TRAIL, and to immune effector cell dependent killing of autologous primary tumors., Results: As many as 40% of primary human tumors express TRAILshort by both RNA sequencing and IHC analysis. By ISH, TRAILshort expression is present in tumor cells and not bystander cells. TRAILshort inhibition enhances cancer cell lines sensitivity to TRAIL-dependent killing both in vitro and in immunodeficient xenograft mouse models. Immune effector cells isolated from patients with B-cell malignancies killed more autologous tumor cells in the presence compared with the absence of TRAILshort antibody ( P < 0.05)., Conclusions: These results identify TRAILshort in primary human malignancies, and suggest that TRAILshort blockade can augment the effector function of autologous immune effector cells. See related commentary by de Miguel and Pardo, p. 5546 ., (©2020 American Association for Cancer Research.)

Purpose: T-cell immunoglobulin and ITIM domain (TIGIT), a member of the immune checkpoint family, is important in normal T-cell biology. However, the phenotypical profile and clinical relevance of TIGIT in follicular lymphoma is largely unknown., Experimental Design: Biopsy specimens from a cohort of 82 patients with follicular lymphoma were analyzed using mass cytometry to explore the phenotype and biological and clinical significance of TIGIT + T cells., Results: TIGIT is highly expressed on intratumoral T cells and its expression alters T-cell phenotype in follicular lymphoma. TIGIT is abundantly expressed on T reg cells, resulting in an enhanced suppressive property. TIGIT expression on non-T reg /T FH T cells defines a population that exhibits an exhausted phenotype. Clinically, increased numbers of TIGIT + T cells are associated with inferior patient outcomes and poor survival. We observe that anti-PD-1 therapy with pembrolizumab alters the phenotype of TIGIT + T subsets and identifies a role for CD28 expression on TIGIT + T cells in treatment response., Conclusions: The current study provides a comprehensive analysis of the phenotypic profile of intratumoral TIGIT + T subsets and their prognostic relevance in follicular lymphoma. Inhibition of TIGIT signaling may be an additional mechanism to prevent T-cell suppression and exhaustion in B-cell lymphoma., (©2020 American Association for Cancer Research.)

Metabolic reprogramming is a hallmark of cancer cells. In Waldenstrom Macroglobulinemia (WM), the infiltration of IgM-secreting lymphoplamacytic cells into the bone marrow (BM) could shift the homeostasis of proteins and metabolites towards a permissive niche for tumor growth. Here, we investigated whether alerted metabolic pathways contribute to the pathobiology of WM and whether the cytokine composition of the BM promotes such changes. Metabolomics analysis on WM patients and normal donors' serum samples revealed a total of 75 metabolites that were significantly altered between two groups. While these metabolites belonged to amino acids, glucose, glutathione and lipid metabolism pathways, the highest number of the differentially expressed metabolites belonged to glutathione metabolism. Proteomics analysis and immunohistochemical staining both confirmed the increased protein levels mediating glutathione metabolism, including GCLC, MT1X, QPCT and GPX3. Moreover, treatment with IL-6 and IL-21, cytokines that induce WM cell proliferation and IgM secretion, increased gene expression of the amino acid transporters mediating glutathione metabolism, including ASCT2, SLC7A11 and 4F2HC, indicating that cytokines in the WM BM could modulate glutathione metabolism. Glutathione synthesis inhibition using Buthionine sulphoximine (BSO) significantly reduced WM cells proliferation in vitro, accompanied with decreased NFκB-p65 and MAPK-p38 phosphorylation. Moreover, BSO treatment significantly reduced the tumor growth rate in a WM xenograft model, further highlighting the role of glutathione metabolism in promoting tumor growth and proliferation. In summary, our data highlight a central role for glutathione metabolism in WM pathobiology and indicate that intervening with the metabolic processes could be a potential therapy for WM patients., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Histological transformation in Waldenström macroglobulinemia (WM) is an uncommon complication, with limited data, particularly regarding the impact of MYD88 L265P mutation on transformation. We examined risk factors and outcomes associated with transformation in WM, highlighting the role of MYD88 L265P mutation. Patients with WM seen at Mayo Clinic, Rochester, USA and University Hospital of Reims, France, between 01/01/1996 and December 31, 2017 were included; 50 (4.3%) of 1147 patients transformed to a high-grade lymphoma, with median time-to-transformation of 4.5 (range 0-21) years in the transformed cohort. The MYD88 L265P mutation status was known in 435/1147 (38%) patients (406 with non-transformed WM and 29 patients in transformed cohort). On multivariate analysis, MYD88 WT status alone was an independent predictor of transformation (odds ratio, 7[95%CI: 2.1-23]; P = .003). Additionally, the MYD88 WT status was independently associated with shorter time-to-transformation (HR 7.9 [95%CI: 2.3-27; P = .001]), with a 5-year transformation rate of 16% for MYD88 WT vs 2.8% with MYD88 L265P mutated patients. Patients with transformation demonstrated a significant increase in risk of death compared to patients who did not transform (HR 5.075; 95%CI: 3.8-6.8; P < .001). In conclusion, the MYD88 WT status is an independent predictor of transformation and associated with a shorter time-to-transformation. Additionally, transformation conferred an inferior overall survival in patients with WM., (© 2019 Wiley Periodicals, Inc.)

Ibrutinib-related data in Waldenström macroglobulinaemia (WM) remain sparse, particularly outside of trials. We report on 80 patients [previously treated, n = 67 (84%), treatment-naïve, n = 13 (16%)] with WM, evaluated consecutively at Mayo Clinic, who received ibrutinib off-study after its approval in 2015 for WM. Overall response rate (ORR) was 91%; major-response rate (MRR) was 78%. The median time to first response and best response was 2·9 [95% confidence interval (CI): 2-4] and 5·7 (95% CI: 4-12) months, respectively. The median follow-up was 19 (95% CI: 14-21) months; 18-month progression-free survival (PFS) was 82%. The median time on therapy was 12·5 (95% CI: 9·3-16·7) months, and the median duration-of-response was 32 (range: 23-32) months. Twenty-five patients (31%) had discontinued therapy at last follow-up (68% due to treatment-related toxicities) and 18% of patients required dose reduction. Fatigue (12%) and atrial-fibrillation (11%) were common non-haematological toxicities. IgM rebound occurred in 36% of patients who abruptly discontinued ibrutinib. Following ibrutinib discontinuation, 84% of patients received subsequent treatment, achieving an ORR of 57% and MRR of 50%. The median PFS from commencement of subsequent salvage therapy was 18 months. Ibrutinib therapy, outside of clinical trials, is effective in WM, but is associated with toxicities and challenges, including IgM rebound and a high drug discontinuation rate for reasons other than disease progression., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

Signal regulatory protein-α (SIRPα) is a key member of the "do-not-eat-me" signaling pathway, but its biological role and clinical relevance in B-cell NHL is relatively unknown. Using biopsy specimens from follicular lymphoma (FL), we identified three subsets (CD14 + SIRPα hi , CD14 - SIRPα low , and CD14 - SIRPα neg ) of monocyte/macrophages (Mo/MΦ) based on CD14 and SIRPα expression. CD14 + SIRPα hi cells expressed common Mo/MΦ markers; exhibited characteristic differentiation, migration, and phagocytosis; and suppressed T-cell function. CD14 - SIRPα low cells expressed fewer typical Mo/MΦ markers; migrated less and phagocytosed tumor cells less efficiently; and stimulated rather than suppressed T-cell function. Interestingly, the CD14 - SIRPα neg subset expressed distinct Mo/MΦ markers compared to the other two subsets; had limited ability to migrate and phagocytose; but stimulated T-cell function. When using SIRPα-Fc to block the interaction between SIRPα and CD47, alone or in combination with rituximab, phagocytosis of tumor cells was differentially increased in the three Mo/MΦ subsets. Clinically, increased numbers of CD14 + SIRPα hi cells were associated with an inferior survival in FL. In contrast, increased numbers of the CD14 - SIRPα low subset appeared to correlate with a better survival. Taken together, our results show that SIRPα expression delineates unique subsets of intratumoral Mo/MΦs with differing prognostic importance.

Epidemiologic studies show an increased risk of non-Hodgkin lymphoma (NHL) in patients with autoimmune disease (AD), due to a combination of shared environmental factors and/or genetic factors, or a causative cascade: chronic inflammation/antigen-stimulation in one disease leads to another. Here we assess shared genetic risk in genome-wide-association-studies (GWAS). Secondary analysis of GWAS of NHL subtypes (chronic lymphocytic leukemia, diffuse large B-cell lymphoma, follicular lymphoma, and marginal zone lymphoma) and ADs (rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis). Shared genetic risk was assessed by (a) description of regional genetic of overlap, (b) polygenic risk score (PRS), (c)"diseasome", (d)meta-analysis. Descriptive analysis revealed few shared genetic factors between each AD and each NHL subtype. The PRS of ADs were not increased in NHL patients (nor vice versa). In the diseasome, NHLs shared more genetic etiology with ADs than solid cancers (p = .0041). A meta-analysis (combing AD with NHL) implicated genes of apoptosis and telomere length. This GWAS-based analysis four NHL subtypes and three ADs revealed few weakly-associated shared loci, explaining little total risk. This suggests common genetic variation, as assessed by GWAS in these sample sizes, may not be the primary explanation for the link between these ADs and NHLs., (© 2019 Wiley Periodicals, Inc.)

Copy number alterations (CNAs) of 9p24.1 occur frequently in Hodgkin lymphoma, primary mediastinal large B-cell lymphoma (PMBCL), primary central nervous system lymphoma, and primary testicular lymphoma, resulting in overexpression of PD-L1 and sensitivity to PD-1 blockade-based immunotherapy. While 9p24.1 CNA was also reported in diffuse large B-cell lymphoma (DLBCL), little is known about its molecular or clinical significance. In this study, we analyzed the prevalence of 9p24.1 CNA in newly diagnosed DLBCL and examined its association with PD-L1, PD-L2, and JAK2 expression, clinical characteristics, and outcome. We found that 10% of DLBCL cases had CNA of 9p24.1, with 6.5% gains, and 3.5% amplifications. Only the cases with a 9p24.1 amplification had high levels of PD-L1, PD-L2, and JAK2 expression. Gains or amplifications of 9p24.1 were associated with a younger age and the ABC/non-GCB subtype. Compared with DLBCL cases without 9p24.1 CNA, the cases with a 9p24.1 amplification had a trend of better event-free survival. Furthermore, the amplification cases had a gene expression and mutation profile similar to those of PMBCL. Our data suggest that amplification of 9p24.1 identifies a unique subset of DLBCL with clinical and molecular features resembling PMBCL that may be amenable to PD-1 blockade-based immunotherapy.

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

Indolent B-cell lymphomas other than follicular lymphoma account for up to 10% of all B-cell neoplasms. While they typically follow a slowly progressive course, some patients experience rapid progression and early mortality. Prognostic scoring systems have not been adopted, hindering the ability of clinicians or researchers to predict outcomes, or risk-stratify patients during clinical trials. To address this, we evaluated the utility of existing prognostic indices and novel, early disease-related outcomes, to predict subsequent long term survival. Baseline characteristics and outcomes data were generated from a longitudinal cohort study that prospectively enrolled 632 patients newly diagnosed with marginal zone lymphoma, lymphoplasmacytic lymphomas, or B-cell lymphomas not otherwise specified, beginning in 2002. The International Prognostic Index (IPI), Follicular Lymphoma International Prognostic Index (FLIPI), and MALT International prognostic index (MALT-IPI) demonstrated c-statistics that ranged from 0.593-0.612 for event-free survival (EFS), and 0.683-0.714 for overall survival (OS). Patients who attained event-free survival at 12 months (EFS12) experienced similar mortality to the US general population (standardized mortality ratio [SMR] 1.19; 95% CI 0.95-1.46). Patients who did not attain EFS12 had subsequent worse morality (SMR 3.14 (95% CI 2.05-4.59). The MALT-IPI demonstrated utility in predicting subsequent long-term outcomes among patients with non-follicular indolent B-cell lymphomas. This index should be used by clinicians giving guidance to patients at the time of initial diagnosis, and risk stratification during clinical studies. The divergent long-term outcomes experienced by patients who do or do not attain EFS12 suggest there exists a subset of patients who harbor high-risk disease. Future research efforts should focus on methods to identify these patients at the time of diagnosis, in order to enable risk-tailored therapy., (© 2019 Wiley Periodicals, Inc.)

Follicular lymphoma (FL) is an indolent B cell malignancy characterized by an extensive but poorly functional T cell infiltrate in the tumor microenvironment. Using mass cytometry, we identified at least 12 subsets of intratumoral CD4 + T cells, 3 of which were unique to FL biopsies versus control tissues. Of these subsets, the frequency of naive T cells correlated with improved patient survival. Although total PD-1 + T cell numbers were not associated with patient outcome, specific PD-1 + T cell subpopulations were associated with poor survival. Intratumoral T cells lacking CD27 and CD28 co-stimulatory receptor expression were enriched in FL and correlated with inferior patient outcomes. In vitro models revealed that CD70 + lymphoma cells played an important role in expanding this population. Taken together, our mass cytometry results identified CD4 + memory T cell populations that are poorly functional due to loss of co-stimulatory receptor expression and are associated with an inferior survival in FL., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Treatment with programmed death-1 (PD-1) blocking antibodies results in high overall response rates in refractory and relapsed classical Hodgkin lymphoma (cHL) patients, indicating that PD-1/PD-1 ligand interactions are integral to progression of this disease. Given the genetically driven increased PD-L1/2 expression in HL, we hypothesized that reverse signaling through PD-1 ligands may be a potential mechanism contributing to the growth and survival of Hodgkin Reed-Sternberg (HRS) cells in cHL. Our data show that engagement of PD-L1 using an agonistic monoclonal antibody increases cell survival and proliferation and reduces apoptosis in HL cell lines. We show that HL patients have significantly higher serum levels of soluble PD-1 than healthy controls, and find that both membrane-bound and soluble forms of PD-1 are able to induce PD-L1 reverse signaling in HL cell lines. PD-L1 signaling, which is associated with activation of the MAPK pathway and increased mitochondrial oxygen consumption, is reversed by PD-1 blockade. In summary, our data identify inhibition of reverse signaling through PD-L1 as an additional mechanism that accounts for clinical responses to PD-1 blockade in cHL.

Waldenström macroglobulinemia (WM)/lymphoplasmacytic lymphoma (LPL) is a rare, chronic B-cell lymphoma with high heritability. We conduct a two-stage genome-wide association study of WM/LPL in 530 unrelated cases and 4362 controls of European ancestry and identify two high-risk loci associated with WM/LPL at 6p25.3 (rs116446171, near EXOC2 and IRF4; OR = 21.14, 95% CI: 14.40-31.03, P = 1.36 × 10 -54 ) and 14q32.13 (rs117410836, near TCL1; OR = 4.90, 95% CI: 3.45-6.96, P = 8.75 × 10 -19 ). Both risk alleles are observed at a low frequency among controls (~2-3%) and occur in excess in affected cases within families. In silico data suggest that rs116446171 may have functional importance, and in functional studies, we demonstrate increased reporter transcription and proliferation in cells transduced with the 6p25.3 risk allele. Although further studies are needed to fully elucidate underlying biological mechanisms, together these loci explain 4% of the familial risk and provide insights into genetic susceptibility to this malignancy.

MYD88 mutations are one of the most recurrent mutations in hematologic malignancies. However, recent mouse models suggest that MYD88 L265P alone may not be sufficient to induce tumor formation. Interplay between MYD88 L265P and other genetic events is further supported by the fact that TNFAIP3 (A20) inactivation often accompanies MYD88 L265P . However, we are still lacking information about the consequence of MYD88 L265P in combination with TNFAIP3 loss in human B cell lymphoma. Review of our genetic data on diffuse large B cell lymphoma (DLBCL) and Waldenstrom macroglobulinemia (WM), found that a large percentage of DLBCL and WM cases that have a MYD88 mutation also harbor a TNFAIP3 loss, 55% DLBCL and 28% of WM, respectively. To mimic this combination of genetic events, we used genomic editing technology to knock out TNFAIP3 in MYD88 L265P non-Hodgkin's lymphoma (NHL) cell lines. Loss of A20 expression resulted in increased NF-κB and p38 activity leading to upregulation of the NF-κB target genes BCL2 and MYC. Furthermore, we detected the increased production of IL-6 and CXCL10 which led to an upregulation of the JAK/STAT pathway. Overall, these results suggest that MYD88 L265P signaling can be enhanced by a second genetic alteration in TNFAIP3 and highlights a potential opportunity for therapeutic targeting.

We evaluated the association of Human Pegivirus (HPgV) viraemia with risk of developing lymphoma, overall and by major subtypes. Because this virus has also been associated with better prognosis in the setting of co-infection with human immunodeficiency virus, we further assessed the association of HPgV with prognosis. We used risk factor data and banked plasma samples from 2094 lymphoma cases newly diagnosed between 2002 and 2009 and 1572 frequency-matched controls. Plasma samples were tested for HPgV RNA by reverse transcription polymerase chain reaction (RT-PCR), and those with RNA concentrations <5000 genome equivalents/ml were confirmed using nested RT-PCR methods. To assess the role of HPgV in lymphoma prognosis, we used 2948 cases from a cohort study of newly diagnosed lymphoma patients (included all cases from the case-control study). There was a positive association of HPgV viraemia with risk of lymphoma overall (Odds ratio = 2·14; 95% confidence interval [CI] 1·63-2·80; P < 0·0001), and for all major subtypes except Hodgkin lymphoma and chronic lymphocytic leukaemia/small lymphocytic lymphoma, and this was not confounded by other lymphoma risk factors. In contrast, there was no association of HPgV viraemia with event-free survival (Hazard ratio [HR] = 1·00; 95% CI 0·85-1·18) or overall survival (HR = 0·97; 95% CI 0·79-1·20) for lymphoma overall, or any of the subtypes. These data support the hypothesis for a role of HPgV in the aetiology of multiple lymphoma subtypes., (© 2018 British Society for Haematology and John Wiley & Sons Ltd.)

Although immune checkpoint molecules regulate the progression of certain cancers, their significance in malignant development of Waldenstrom macroglobulinemia (WM), an incurable low-grade B-cell lymphoma, remains unknown. Recently, cytokines in the bone marrow (BM) microenvironment are shown to contribute to the pathobiology of WM. Here, we investigated the impact of cytokines, including interleukin-6 (IL-6) and IL-21, on immune regulation and particularly on the programmed death-1 (PD-1) and its ligands PD-L1 and PD-L2. We showed that IL-21, interferon γ, and IL-6 significantly induced PD-L1 and PD-L2 gene expression in WM cell lines. Increased PD-L1 and PD-L2 messenger RNA was also detected in patients' BM cells. Patients' nonmalignant BM cells, including T cells and monocytes, showed increased PD-L1, but minimal or undetectable PD-L2 surface expression. There was also very modest PD-L1 and PD-L2 surface expression by malignant WM cells, suggesting that ligands are cleaved from the cell surface. Levels of soluble ligands were higher in patients' BM plasma and blood serum than controls. Furthermore, IL-21 and IL-6 increased secreted PD-L1 in the culture media of WM cell lines, implying that elevated levels of soluble PD-1 ligands are cytokine mediated. Soluble PD-1 ligands reduced T-cell proliferation, phosphorylated extracellular signal-regulated kinase and cyclin A levels, mitochondrial adenosine triphosphate production, and spare respiratory capacity. In conclusion, we identify that soluble PD-1 ligands are elevated in WM patients and, in addition to surface-bound ligands in WM BM, could regulate T-cell function. Given the capability of secreted forms to be bioactive at distant sites, soluble PD-1 ligands have the potential to promote disease progression in WM., (© 2018 by The American Society of Hematology.)