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Peripheral T-cell lymphoma (PTCL) is a heterogeneous category, and many cases are unclassifiable and designated as PTCL-not otherwise specified (PTCL-NOS). Gene expression profiling (GEP) has delineated two prognostic subtypes within PTCL-NOS, PTCL-TBX21, and PTCL-GATA3, characterized by distinctive transcriptomes and a different prognosis. To further evaluate the pathologic features of these subgroups, 101 PTCL cases that did not meet specific criteria for well-defined T-cell lymphoma entities underwent detailed pathologic, immunophenotypic (including T FH biomarkers) and GEP analyses, separating them into PTCL-NOS (n=63) and PTCL-TFH (a.k.a. nodal PTCL-TFH, NOS, and TFH lymphoma, NOS) (n=38). PTCL-NOS cases were further categorized into PTCL-GATA3 (n=22; 34%) and PTCL-TBX21 (n=41; 66%), and a significant association (p < 0.02) with overall survival (OS) was reaffirmed. Histopathological assessment showed PTCL-GATA3 cases were characterized by monotonous medium-sized or large transformed cells with a minimal tumor microenvironment (TME) compared to PTCL-TBX21 cases, which consisted of pleomorphic cells in a polymorphous TME (p < 0.05). GEP analysis validated these TME distinctions. Immunophenotypic analysis showed that PTCL-GATA3 cases were predominantly CD4+CD8- and associated with significantly higher LEF1, MYC, and CD30 expression (p < 0.05). PTCL-TBX21 displayed a more diverse biomarker profile with two subgroups: one expressing cytotoxic antigens and enriched in CD8+CD4- or CD8-CD4- phenotype, and another lacking cytotoxic markers but showing a CD4+CD8- phenotype with increased ICOS expression, but devoid of other T FH markers. The PTCL-TFH cases correlated with an angioimmunoblastic T-cell lymphoma (AITL) gene signature, had more EBER-positive cells than the PTCL-GATA3 and PTCL-TBX21 cases, and a subset had some morphologic features of AITL (p < 0.01). This study highlights the unique morphologic and phenotypic variations within the newly-identified PTCL subtypes and should enable more precise diagnosis and tailored therapeutic strategies in the future., (Copyright © 2024. Published by Elsevier Inc.)

Recent advances in Rosai-Dorfman-Destombes disease (RDD), notably molecular testing, targeted therapy, and PET-CT imaging, hold promise for better recognition and improved outcomes. This study presents patients diagnosed and treated in a "real world" setting, where navigating limited resources must be considered. This retrospective single-center review includes 15 adult patients diagnosed with RDD at Vancouver General Hospital between November 2015 and October 2023. The cohort comprised five males and ten females with a median age 53 years (range 19-80 years). All 15 patients had extra-nodal disease; 11 patients exclusively had extra-nodal disease, and four patients also had lymph node involvement. Seven patients had tissue next-generation sequencing, identifying MAP2K1 mutations in four cases and a KRAS p.K117N mutation in one case that was treated with targeted therapy using trametinib. PET-CT was used for disease staging in four cases. Six patients with refractory disease tolerated lenalidomide and dexamethasone without significant toxicity; three patients achieved complete response, and three had partial response. This study highlights RDD's diverse extra-nodal manifestations. Lenalidomide combined with dexamethasone is an effective and well-tolerated treatment option for select patients, especially those with refractory disease. Broad utilization of NGS and PET-CT can positively influence management decisions., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

High-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2), or 'double-hit lymphoma,' has been associated with a high risk of central nervous system (CNS) relapse. However, historic estimates are impacted by selection bias. We report CNS relapse rates associated with HGBCL-DH-BCL2 from a population-based cohort with complete fluorescence in situ hybridization testing, as well as diffuse large B-cell lymphoma morphology (DLBCL) tumors expressing the dark-zone gene expression signature (DZsig), which was originally derived from HGBCL-DH-BCL2. The 2-year CNS relapse risk in HGBCL-DH-BCL2 was 6.8%. CNS relapses were early, predominantly leptomeningeal (73%) and co-occurred with systemic relapse (64%). High-risk CNS International Prognostic Index (CNS-IPI) and concordant bone marrow involvement were associated with an elevated CNS relapse risk in HGBCL-DH-BCL2. The 'refined cell of origin' classification assigned 20% of DLBCL morphology tumors with germinal center B-cell-like phenotype (GCB-DLBCL) into a distinct subgroup based on DZsig expression (DZsig+). CNS relapse risk in DZsig+ (2-year: 6.4%) was independent of HGBCL-DH-BCL2 status and was further stratified by the CNS-IPI. CNS relapse in DZsig-negative GCB-DLBCL was rare (2-year risk 1.4%; P=.04 versus DZsig+) and exclusively parenchymal. Altogether, the CNS relapse risk in HGBCL-DH-BCL2 is lower than previously reported and DZsig refines risk stratification in GCB-DLBCL., (Copyright © 2024 American Society of Hematology.)

Abstract: Fluorescence in situ hybridization (FISH) using break-apart probes is recommended for identifying high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). Unbalanced MYC break-apart patterns, in which the red or green signal is lost, are commonly reported as an equivocal result by clinical laboratories. In a cohort of 297 HGBCL-DH-BCL2, 13% of tumors had unbalanced MYC break-apart patterns with loss of red (LR; 2%) or loss of green (LG; 11%) signal. To determine the significance of these patterns, MYC rearrangements were characterized by sequencing in 130 HGBCL-DH-BCL2, including 3 LR and 14 LG tumors. A MYC rearrangement was identified for 71% of tumors with LR or LG patterns, with the majority involving immunoglobulin loci or other recurrent MYC rearrangement partners. The architecture of these rearrangements consistently preserved the rearranged MYC allele, with the MYC gene predicted to be on the derivative chromosome containing the signal that is still present in nearly all cases. MYC protein expression, MYC messenger RNA expression, and the proportion of tumors expressing the dark-zone signature was not significantly different between balanced and unbalanced groups. These results support a recommendation that unbalanced MYC break-apart FISH patterns be reported as positive for MYC rearrangement in the context of diagnosing HGBCL-DH-BCL2., (© 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.)

Abstract: Rearrangements that place the oncogenes MYC, BCL2, or BCL6 adjacent to superenhancers are common in mature B-cell lymphomas. Lymphomas with diffuse large B-cell lymphoma (DLBCL) or high-grade morphology with both MYC and BCL2 rearrangements are classified as high-grade B-cell lymphoma with MYC and BCL2 rearrangements ("double hit"; HGBCL-DH-BCL2) and are associated with aggressive disease and poor outcomes. Although it is established that MYC rearrangements involving immunoglobulin (IG) loci are associated with inferior outcomes relative to those involving other non-IG superenhancers, the frequency of and mechanisms driving IG vs non-IG MYC rearrangements have not been elucidated. Here, we used custom targeted capture and/or whole-genome sequencing to characterize oncogene rearrangements across 883 mature B-cell lymphomas including Burkitt lymphoma, follicular lymphoma, DLBCL, and HGBCL-DH-BCL2 tumors. We demonstrate that, although BCL2 rearrangement topology is consistent across entities, HGBCL-DH-BCL2 have distinct MYC rearrangement architecture relative to tumors with single MYC rearrangements or with both MYC and BCL6 rearrangements (HGBCL-DH-BCL6), including both a higher frequency of non-IG rearrangements and different architecture of MYC::IGH rearrangements. The distinct MYC rearrangement patterns in HGBCL-DH-BCL2 occur on the background of high levels of somatic hypermutation across MYC partner loci in HGBCL-DH-BCL2, creating more opportunity to form these rearrangements. Furthermore, because 1 IGH allele is already disrupted by the existing BCL2 rearrangement, the MYC rearrangement architecture in HGBCL-DH-BCL2 likely reflects selective pressure to preserve both BCL2 and B-cell receptor expression. These data provide new mechanistic explanations for the distinct patterns of MYC rearrangements observed across different lymphoma entities.

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3 , a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL., Competing Interests: Competing interests statement:C.S. has performed consultancy for Bayer. D.W.S. has served as a consultant for Abbvie, AstraZenenca, Incyte, and Janssen. C.S. and D.W.S. are named inventors on a patent filed by the National Cancer Institute “Methods for determining lymphoma type.” The remaining authors declare no competing financial interests.

Purpose: Diffuse large B-cell lymphoma (DLBCL) is cured in more than 60% of patients, but outcomes remain poor for patients experiencing disease progression or relapse (refractory or relapsed DLBCL [rrDLBCL]), particularly if these events occur early. Although previous studies examining cohorts of rrDLBCL have identified features that are enriched at relapse, few have directly compared serial biopsies to uncover biological and evolutionary dynamics driving rrDLBCL. Here, we sought to confirm the relationship between relapse timing and outcomes after second-line (immuno)chemotherapy and determine the evolutionary dynamics that underpin that relationship., Patients and Methods: Outcomes were examined in a population-based cohort of 221 patients with DLBCL who experienced progression/relapse after frontline treatment and were treated with second-line (immuno)chemotherapy with an intention-to-treat with autologous stem-cell transplantation (ASCT). Serial DLBCL biopsies from a partially overlapping cohort of 129 patients underwent molecular characterization, including whole-genome or whole-exome sequencing in 73 patients., Results: Outcomes to second-line therapy and ASCT are superior for late relapse (>2 years postdiagnosis) versus primary refractory (<9 months) or early relapse (9-24 months). Diagnostic and relapse biopsies were mostly concordant for cell-of-origin classification and genetics-based subgroup. Despite this concordance, the number of mutations exclusive to each biopsy increased with time since diagnosis, and late relapses shared few mutations with their diagnostic counterpart, demonstrating a branching evolution pattern. In patients with highly divergent tumors, many of the same genes acquired new mutations independently in each tumor, suggesting that the earliest mutations in a shared precursor cell constrain tumor evolution toward the same genetics-based subgroups at both diagnosis and relapse., Conclusion: These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naïve disease and have implications for optimal patient management.

Follicular lymphoma (FL) accounts for ∼20% of all new lymphoma cases. Increases in cytological grade are a feature of the clinical progression of this malignancy, and eventual histologic transformation (HT) to the aggressive diffuse large B-cell lymphoma (DLBCL) occurs in up to 15% of patients. Clinical or genetic features to predict the risk and timing of HT have not been described comprehensively. In this study, we analyzed whole-genome sequencing data from 423 patients to compare the protein coding and noncoding mutation landscapes of untransformed FL, transformed FL, and de novo DLBCL. This revealed 2 genetically distinct subgroups of FL, which we have named DLBCL-like (dFL) and constrained FL (cFL). Each subgroup has distinguishing mutational patterns, aberrant somatic hypermutation rates, and biological and clinical characteristics. We implemented a machine learning-derived classification approach to stratify patients with FL into cFL and dFL subgroups based on their genomic features. Using separate validation cohorts, we demonstrate that cFL status, whether assigned with this full classifier or a single-gene approximation, is associated with a reduced rate of HT. This implies distinct biological features of cFL that constrain its evolution, and we highlight the potential for this classification to predict HT from genetic features present at diagnosis., (© 2023 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.)

Objectives: Multiple case reports describe Kikuchi-Fujimoto disease (KFD) following COVID-19 vaccination, but the true nature of this phenomenon is unknown. The purpose of this study was to further assess the relationship between KFD and COVID-19 vaccination at the population level., Methods: Confirmed KFD cases from January 2018 to April 2022 were identified from provincial pathology archives and analyzed in the context of vaccination statistics from public health resources., Results: Our statistical models provide evidence of a temporal association between KFD and both antecedent COVID-19 vaccine administration as well as age-stratified vaccination rates. Eight new cases of plausible COVID-19 vaccine-associated KFD are presented, collectively exhibiting clinical and pathologic features that overlap substantially with those of idiopathic KFD., Conclusions: Our findings indicate that KFD is observed in association with COVID-19 vaccination and suggest that mechanistic studies are warranted., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society for Clinical Pathology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Lymphoma is a clinically and biologically heterogeneous disease. Next-generation sequencing (NGS) has expanded our understanding of this heterogeneity at the genetic level, refining disease classification, defining new entities, and providing additional information that can be used in diagnosis and management. This review highlights some of the NGS findings in lymphoma and how they can be used as genetic biomarkers to aid diagnosis and prognosis and guide therapy., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) underlies the variable outcomes achieved with immunochemotherapy. However, outcomes of gene expression profiling (GEP)-defined molecular subgroups in a real-world DLBCL population remain unknown. Here we examined the prevalence and outcomes of molecular subgroups in an unselected population of 1149 patients with de novo DLBCL in British Columbia, Canada. Evaluable biopsies were profiled by fluorescence in situ hybridization (FISH), immunohistochemistry, and digital GEP to assign cell-of-origin and the so-called "double-hit signature" (DHITsig)-a signature originally described as being characteristic for high-grade B-cell lymphoma with MYC and BCL2 rearrangements (HGBCL-DH-BCL2). DHITsig was expressed in 21% of 431 germinal center B-cell-like (GCB)-DLBCL and all 55 Burkitt lymphomas examined. Reflecting this latter finding, DHITsig has been renamed the "dark zone signature" (DZsig). DZsigpos-DLBCL, non-DZsigpos GCB-DLBCL and activated B-cell-like (ABC)-DLBCL were associated with a 2 year overall survival of 57%, 89%, and 71%, respectively. 62% of DZsigpos tumors were negative for HGBCL-DH-BCL2 by FISH, but were associated with outcomes similar to HGBCL-DH-BCL2. A small group of HGBCL-DH-BCL2 that lacked DZsig expression had different molecular features compared with DZsig-expressing HGBCL-DH-BCL2 and were associated with favorable outcomes comparable to DLBCL, not otherwise specified. DZsigpos and ABC-DLBCL had a shorter diagnosis-to-treatment interval (DTI) than GCB-DLBCL, with this metric being associated with outcome. In conclusion, DZsig expression extends beyond HGBCL-DH-BCL2 and captures a poor-prognosis DLBCL subgroup with short DTI, including patients unidentifiable by routine FISH testing, that should be considered for treatment intensification or novel therapies in prospective trials., (© 2023 by The American Society of Hematology.)

Context.—: Although CD30 testing is an established tool in the diagnostic workup of lymphomas, it is also emerging as a predictive biomarker that informs treatment. The current definition of CD30 positivity by immunohistochemistry is descriptive and based on reactivity in lymphomas that are defined by their universal strong expression of CD30, rather than any established threshold. Challenges include inconsistencies with preanalytic variables, tissue processing, pathologist readout, and with the pathologist and oncologist interpretation of reported results., Objective.—: To develop and propose general best practice recommendations for reporting CD30 expression by immunohistochemistry in lymphoma biopsies to harmonize practices across institutions and facilitate assessment of its significance in clinical decision-making., Design.—: Following literature review and group discussion, the panel of 14 academic hematopathologists and 2 clinical/academic hematologists/oncologists divided into 3 working groups. Each working group was tasked with assessing CD30 testing by immunohistochemistry, CD30 expression readout, or CD30 expression interpretation., Results.—: Panel recommendations were reviewed and discussed. An online survey was conducted to confirm the consensus recommendations., Conclusions.—: CD30 immunohistochemistry is required for all patients in whom classic Hodgkin lymphoma and any lymphoma within the spectrum of peripheral T-cell lymphoma are differential diagnostic considerations. The panel reinforced and summarized that immunohistochemistry is the preferred methodology and any degree of CD30 expression should be reported. For diagnostic purposes, the interpretation of CD30 expression should follow published guidelines. To inform therapeutic decisions, report estimated percent positive expression in tumor cells (or total cells where applicable) and record descriptively if nontumor cells are positive., (© 2023 College of American Pathologists.)

Outcomes in older adults with classic Hodgkin lymphoma (cHL) have traditionally been poor, in part, related to poor tolerance to standard chemotherapy. Herein, we evaluated the survival of patients with cHL aged ≥60 years in British Columbia in a population-based analysis. From 1961 to 2019, 744 patients with newly diagnosed cHL were identified. With a median follow-up of 9 years, 5-year disease-specific survival (DSS) and overall survival (OS) have improved by decade comparison (both P < .001), remaining stable in the past 20 years (DSS, P = .35; OS, P = .26). In the modern management era (2000-present), 361 of 401 patients (90%) received active therapy for cHL and had a 5-year OS of 60%. For those who received curative-intent therapy (n = 327), the 5-year progression-free survival (PFS), OS, and DSS were 60%, 65%, and 76%, respectively, and estimates were superior in those who were 60 to 69 years of age (72%, 77%, and 83%, respectively) compared with those who were 70 to 79 years of age (54%, 57%, and 70%, respectively) and ≥80 years of age (28%, 39%, and 63%, respectively) (P < .05 for all). Overall, pulmonary toxicity occurred in 58 of 279 patients (21%) treated with bleomycin, with 22 of 58 (38%) occurring after cycles 1 or 2, accounting for 8 of 20 (40%) treatment-related deaths. Outcomes in older adults with cHL have improved in recent decades; however, they remain poor for those aged ≥70 years, even in the modern treatment era. Furthermore, treatment-related toxicity remains a significant concern and use of bleomycin should be avoided in most patients., (© 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.)

Plasmablastic lymphoma (PBL) is an aggressive and rare subtype of non-Hodgkin lymphoma with no standard-of-care therapy. We reviewed all patients diagnosed with histologically confirmed PBL in British Columbia, Canada between 1997 and 2019. Overall, 42 patients were identified, including 15 (36%) positive for HIV and nine (21%) on chronic immunosuppression. Curative-intent treatment consisting primarily of cyclophosphamide, doxorubicin, vincristine and prednisone was administered to 31 patients, of which 74% achieved response, however 61% relapsed after a median of 7.5 months. At a median follow-up of eight years for the whole cohort, five-year progression-free survival (PFS) and overall survival (OS) were 18% [95% confidence interval (CI): 6%, 30%] and 22% (95% CI: 8%, 36%) with median eight and 15 months respectively. There were no differences in relapse rate (p = 0.962), PFS (p = 0.228) or OS (p = 0.340) according to immune status. For those treated with curative intent, five-year PFS and OS were 24% (95% CI: 8%, 40%) and 31% (95% CI: 13%, 49%) with median 18 and 27 months respectively. In this population-based cohort of PBL patients spanning 20 years, survival outcomes were poor. Ultimately, further research is needed to develop more effective treatment strategies and to improve survival for patients., (© 2022 British Society for Haematology and John Wiley & Sons Ltd.)

Activated B cell-like diffuse large B-cell lymphomas (ABC-DLBCL) have unfavorable outcomes and chronic activation of CARD11-BCL10-MALT1 (CBM) signal amplification complexes that form due to polymerization of BCL10 subunits, which is affected by recurrent somatic mutations in ABC-DLBCLs. Herein, we show that BCL10 mutants fall into at least two functionally distinct classes: missense mutations of the BCL10 CARD domain and truncation of its C-terminal tail. Truncating mutations abrogated a motif through which MALT1 inhibits BCL10 polymerization, trapping MALT1 in its activated filament-bound state. CARD missense mutations enhanced BCL10 filament formation, forming glutamine network structures that stabilize BCL10 filaments. Mutant forms of BCL10 were less dependent on upstream CARD11 activation and thus manifested resistance to BTK inhibitors, whereas BCL10 truncating but not CARD mutants were hypersensitive to MALT1 inhibitors. Therefore, BCL10 mutations are potential biomarkers for BTK inhibitor resistance in ABC-DLBCL, and further precision can be achieved by selecting therapy based on specific biochemical effects of distinct mutation classes., Significance: ABC-DLBCLs feature frequent mutations of signaling mediators that converge on the CBM complex. We use structure-function approaches to reveal that BCL10 mutations fall into two distinct biochemical classes. Both classes confer resistance to BTK inhibitors, whereas BCL10 truncations confer hyperresponsiveness to MALT1 inhibitors, providing a road map for precision therapies in ABC-DLBCLs. See related commentary by Phelan and Oellerich, p. 1844. This article is highlighted in the In This Issue feature, p. 1825., (©2022 The Authors; Published by the American Association for Cancer Research.)

A 54-Year-Old Woman with Cutaneous NodulesA 54-year-old woman presented with chronic cutaneous nodules and plaques. How do you approach the evaluation, and what is the diagnosis?

Objectives: Intravascular large B-cell lymphomas (IVLBCLs) are rare extranodal LBCLs in which relapse is relatively frequent. We sought to further characterize potential immune escape mechanisms in IVLBCLs that newer therapies can exploit., Methods: A series of 33 IVLBCLs were evaluated for programmed cell death ligand 1 (PD-L1) and PD-L2 expression by immunohistochemistry (IHC), chromosomal alterations (CAs) in the PDL1/PDL2 locus by fluorescence in situ hybridization, and loss of major histocompatibility complex (MHC) class I and II expression by IHC., Results: Cases were subclassified as classical (n = 22) or hemophagocytic syndrome (HPS)-associated (n = 11) variants. A total of 12 cases (39%; n = 12/31) expressed PD-L1 and/or PD-L2. CAs were seen in 7 cases (7/29 [24%]) and included gains, amplifications, and rearrangements. CAs in classical variant cases (24%; n = 5/21) included gains (n =1), gains with concurrent rearrangements (n = 2), and amplifications (n = 2). The 2 HPS-associated variant cases with CAs (25%; n = 2/8) both showed amplification, including 1 case with a concurrent rearrangement. A majority of cases with CAs (71%; n = 5/7) were PD-L1/PD-L2 IHC positive. Among PD-L1/PD-L2 IHC-positive cases, 45% harbored a CA. Loss of MHC class I and/or class II was seen in 27% (n = 9/33) of cases., Conclusions: Altogether, our data show that 65% (n = 20/31) of IVLBCLs may exploit immune evasion strategies through PD-L1/PD-L2 expression or downregulation of MHC proteins., (© American Society for Clinical Pathology, 2021. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Purpose: Peripheral T-cell lymphoma (PTCL) is a heterogeneous group of non-Hodgkin lymphomas with aggressive clinical behavior. We performed comprehensive miRNA profiling in PTCLs and corresponding normal CD4 + Th1/2 and TFH-like polarized subsets to elucidate the role of miRNAs in T-cell lymphomagenesis., Experimental Design: We used nCounter (NanoString Inc) for miRNA profiling and validated using Taqman qRT-PCR (Applied Biosystems, Inc). Normal CD4 + T cells were polarized into effector Th subsets using signature cytokines, and miRNA significance was revealed using functional experiments., Results: Effector Th subsets showed distinct miRNA expression with corresponding transcription factor expression (e.g., BCL6/miR-19b, -106, -30d, -26b, in IL21-polarized; GATA3/miR-155, miR-337 in Th2-polarized; and TBX21/miR-181a, -331-3p in Th1-polarized cells). Integration of miRNA signatures suggested activation of TCR and PI3K signaling in IL21-polarized cells, ERK signaling in Th1-polarized cells, and AKT-mTOR signaling in Th2-polarized cells, validated at protein level. In neoplastic counterparts, distinctive miRNAs were identified and confirmed in an independent cohort. Integrative miRNA-mRNA analysis identified a decrease in target transcript abundance leading to deregulation of sphingolipid and Wnt signaling and epigenetic dysregulation in angioimmunoblastic T-cell lymphoma (AITL), while ERK, MAPK, and cell cycle were identified in PTCL subsets, and decreased target transcript abundance was validated in an independent cohort. Elevated expression of miRNAs (miR-126-3p, miR-145-5p) in AITL was associated with poor clinical outcome. In silico and experimental validation suggest two targets (miR-126→ SIPR2 and miR-145 → ROCK1) resulting in reduced RhoA-GTPase activity and T-B-cell interaction., Conclusions: Unique miRNAs and deregulated oncogenic pathways are associated with PTCL subtypes. Upregulated miRNA-126-3p and miR-145-5p expression regulate RhoA-GTPase and inhibit T-cell migration, crucial for AITL pathobiology., (©2021 American Association for Cancer Research.)

Richter syndrome (RS), an aggressive lymphoma occurring in the context of chronic lymphocytic leukaemia/small lymphocytic lymphoma, is associated with poor prognosis when treated with conventional immunochemotherapy, therefore, improved treatments are required. Immune checkpoint blockade has shown efficacy in some B-cell malignancies and modest responses in early clinical trials for RS. We investigated the immune checkpoint profile of RS as a basis to inform rational therapeutic investigations in RS. Formalin-fixed, paraffin-embedded biopsies of RS (n = 19), de novo diffuse large B-cell lymphoma (DLBCL; n = 58), transformed indolent lymphomas (follicular [tFL], n = 16; marginal zone [tMZL], n = 24) and non-transformed small lymphocytic lymphoma (SLL; n = 15) underwent gene expression profiling using the NanoString Human Immunology panel. Copy number assessment was performed using next-generation sequencing. Immunohistochemistry (IHC) for LAG3 and PD-1 was performed. LAG3 gene expression was higher in RS compared to DLBCL (P = 0·0002, log2FC 1·96), tFL (P < 0·0001, log2FC 2·61), tMZL (P = 0·0004, log2FC 1·79) and SLL (P = 0·0057, log2FC 1·45). LAG3 gene expression correlated with the gene expression of human leukocyte antigen Class I and II, and related immune genes and immune checkpoints. IHC revealed LAG3 protein expression on both malignant RS cells and tumour-infiltrating lymphocytes. Our findings support the investigation of LAG3 inhibition to enhance anti-tumour responses in RS., (© 2021 British Society for Haematology and John Wiley & Sons Ltd.)

Radiotherapy (RT) is typically incorporated into the treatment of limited-stage nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), although it remains unknown whether chemotherapy alone may be suitable in select patients. We evaluated outcomes of limited-stage NLPHL at BC Cancer on the basis of era-specific guidelines: routine RT era, 1995 to 2005 (n = 36), combined modality with 2 cycles of doxorubicin, bleomycin, vinblastine, dacarbazine (ABVD) chemotherapy followed by RT or RT alone; positron emission tomography (PET) era, after 2005 (n = 63), ABVD alone (4 cycles) if the PET scan after the second cycle of ABVD (PET2) is negative, or treatment is changed to RT if PET2 is positive. Median age of patients was 38 years (range, 16-82 years), 73% were male, and 43% had stage II. With a median follow-up of 10.5 years for all patients, 5-year progression-free survival (PFS) was 91% [corrected] and was 97% for overall survival (OS), with no difference by treatment era (PFS, P = .15; [corrected] OS, P = .35). For the 49 patients who had a PET2 scan, 86% were PET negative and 14% were PET positive by Deauville criteria with 5-year PFS rates of 92% and 80% (P = .87) [corrected], respectively. This is the largest study of a PET-adapted approach in NLPHL and supports that ABVD alone may be a viable option in select patients with a negative PET2 scan, with consideration of acute and long-term toxicities., (© 2021 by The American Society of Hematology.)

Fc γ receptor IIB (FcγRIIB) is an inhibitory molecule capable of reducing antibody immunotherapy efficacy. We hypothesized its expression could confer resistance in patients with diffuse large B-cell lymphoma (DLBCL) treated with anti-CD20 monoclonal antibody (mAb) chemoimmunotherapy, with outcomes varying depending on mAb (rituximab [R]/obinutuzumab [G]) because of different mechanisms of action. We evaluated correlates between FCGR2B messenger RNA and/or FcγRIIB protein expression and outcomes in 3 de novo DLBCL discovery cohorts treated with R plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) reported by Arthur, Schmitz, and Reddy, and R-CHOP/G-CHOP-treated patients in the GOYA trial (NCT01287741). In the discovery cohorts, higher FCGR2B expression was associated with significantly shorter progression-free survival (PFS; Arthur: hazard ratio [HR], 1.09; 95% confidence interval [CI], 1.01-1.19; P = .0360; Schmitz: HR, 1.13; 95% CI, 1.02-1.26; P = .0243). Similar results were observed in GOYA with R-CHOP (HR, 1.26; 95% CI, 1.00-1.58; P = .0455), but not G-CHOP (HR, 0.91; 95% CI, 0.69-1.20; P = .50). A nonsignificant trend that high FCGR2B expression favored G-CHOP over R-CHOP was observed (HR, 0.67; 95% CI, 0.44-1.02; P = .0622); however, low FCGR2B expression favored R-CHOP (HR, 1.58; 95% CI, 1.00-2.50; P = .0503). In Arthur and GOYA, FCGR2B expression was associated with tumor FcγRIIB expression; correlating with shorter PFS for R-CHOP (HR, 2.17; 95% CI, 1.04-4.50; P = .0378), but not G-CHOP (HR, 1.37; 95% CI, 0.66-2.87; P = .3997). This effect was independent of established prognostic biomarkers. High FcγRIIB/FCGR2B expression has prognostic value in R-treated patients with DLBCL and may confer differential responsiveness to R-CHOP/G-CHOP., (© 2021 by The American Society of Hematology.)

Primary mediastinal large B-cell lymphoma (PMBL) is a type of aggressive B-cell lymphoma that typically affects young adults, characterized by presence of a bulky anterior mediastinal mass. Lymphomas with gene expression features of PMBL have been described in nonmediastinal sites, raising questions about how these tumors should be classified. Here, we investigated whether these nonmediastinal lymphomas are indeed PMBLs or instead represent a distinct group within diffuse large B-cell lymphoma (DLBCL). From a cohort of 325 de novo DLBCL cases, we identified tumors from patients without evidence of anterior mediastinal involvement that expressed a PMBL expression signature (nm-PMBLsig+; n = 16; 5%). A majority of these tumors expressed MAL and CD23, proteins typically observed in bona fide PMBL (bf-PMBL). Evaluation of clinical features of nm-PMBLsig+ cases revealed close associations with DLBCL, and a majority displayed a germinal center B cell-like cell of origin (GCB). In contrast to patients with bf-PMBL, patients with nm-PMBLsig+ presented at an older age and did not show pleural disease, and bone/bone marrow involvement was observed in 3 cases. However, although clinically distinct from bf-PMBL, nm-PMBLsig+ tumors resembled bf-PMBL at the molecular level, with upregulation of immune response, JAK-STAT, and NF-κB signatures. Mutational analysis revealed frequent somatic gene mutations in SOCS1, IL4R, ITPKB, and STAT6, as well as CD83 and BIRC3, with the latter genes significantly more frequently affected than in GCB DLBCL or bf-PMBL. Our data establish nm-PMBLsig+ lymphomas as a group within DLBCL with distinct phenotypic and genetic features. These findings may have implications for gene expression- and mutation-based subtyping of aggressive B-cell lymphomas and related targeted therapies., (© 2021 by The American Society of Hematology.)

When the World Health Organization defined high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) as a clinical category, rearrangements were the only structural variant (SV) incorporated. An "atypical double-hit" category has been proposed, encompassing tumors with concurrent MYC and BCL2 SVs other than cooccurring translocations (ie, copy number variations [CNVs]). Although the identification of a gene expression signature (DHITsig) shared among tumors harboring MYC and BCL2 rearrangements (HGBL-DH/TH-BCL2) has confirmed a common underlying biology, the biological implication of MYC and BCL2 CNVs requires further elucidation. We performed a comprehensive analysis of MYC and BCL2 SVs, as determined by fluorescent in situ hybridization (FISH), in a cohort of 802 de novo tumors with diffuse large B-cell lymphoma morphology. Although BCL2 CNVs were associated with increased expression, MYC CNVs were not. Furthermore, MYC and BCL2 CNVs, in the context of atypical double-hit, did not confer a similar gene expression profile as HGBL-DH/TH-BCL2. Finally, although MYC immunohistochemistry (IHC) has been proposed as a screening tool for FISH testing, 2 mechanisms were observed that uncoupled MYC rearrangement from IHC positivity: (1) low MYC messenger RNA expression; and (2) false-negative IHC staining mediated by a single-nucleotide polymorphism resulting in an asparagine-to-serine substitution at the 11th amino acid residue of MYC (MYC-N11S). Taken together, these results support the current exclusion of MYC and BCL2 CNVs from HGBL-DH/TH and highlight the ability of a molecular-based classification system to identify tumors with shared biology that FISH and IHC fail to fully capture., (© 2021 by The American Society of Hematology.)

The mutational landscape of gray zone lymphoma (GZL) has not yet been established, and differences from related entities are largely unknown. Here, we studied coding sequence mutations of 50 Epstein-Barr virus (EBV)-negative GZLs and 20 polymorphic EBV+ diffuse large B-cell lymphoma (DLBCL) not otherwise specified (poly-EBV-L) in comparison with classical Hodgkin lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), and DLBCL. Exomes of 21 GZL and 7 poly-EBV-L cases, along with paired constitutional DNA, were analyzed as a discovery cohort, followed by targeted sequencing of 217 genes in an extension cohort of 29 GZL and 13 poly-EBV-L cases. GZL cases with thymic niche involvement (anterior mediastinal mass) exhibited a mutation profile closely resembling cHL and PMBCL, with SOCS1 (45%), B2M (45%), TNFAIP3 (35%), GNA13 (35%), LRRN3 (32%), and NFKBIA (29%) being the most recurrently mutated genes. In contrast, GZL cases without thymic niche involvement (n = 18) had a significantly distinct pattern that was enriched in mutations related to apoptosis defects (TP53 [39%], BCL2 [28%], BIRC6 [22%]) and depleted in GNA13, XPO1, or NF-κB signaling pathway mutations (TNFAIP3, NFKBIE, IKBKB, NFKBIA). They also exhibited more BCL2/BCL6 rearrangements compared with thymic GZL. Poly-EBV-L cases presented a distinct mutational profile, including STAT3 mutations and a significantly lower coding mutation load in comparison with EBV- GZL. Our study highlights characteristic mutational patterns in GZL associated with presentation in the thymic niche, suggesting a common cell of origin and disease evolution overlapping with related anterior mediastinal lymphomas., (© 2021 by The American Society of Hematology.)

Consolidative radiation therapy (RT) for advanced-stage diffuse large B-cell lymphoma (DLBCL) remains controversial, with routine practice continuing to include RT in patients with initial bulky disease or residual masses. Positron emission tomography (PET)-computed tomography is a sensitive modality for detecting the presence of residual disease at the end of treatment (EOT). A PET-guided approach to selectively administering RT has been the policy in British Columbia since 2005. Patients with advanced-stage DLBCL diagnosed from 1 January 2005 to 1 March 2017 and treated with at least 6 cycles of R-CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone plus rituximab), who underwent EOT PET, were included in this analysis. Those with complete metabolic response (PET-negative [PET-NEG]) were observed; those with PET-positive (PET-POS) scans were offered consolidative RT, when feasible. Of the patient records reviewed, 723 were identified, with median follow-up of 4.3 years: 517 (72%) were PET-NEG; 206 (28%) were PET-POS. Time to progression (TTP) and overall survival (OS) at 3 years were 83% vs 56% and 87% vs 64%, in patients with PET-NEG and PET-POS scans, respectively. PET-POS patients with nonprogressing disease treated with consolidative RT (109 and 206; 53%) had outcomes approaching those of PET-NEG patients, with 3-year estimates of 76% and 80% for TTP and OS. PET-NEG patients who had bulky disease (≥10 cm) at diagnosis had outcomes indistinguishable from those without bulk, despite the omission of RT. These data suggest that patients with advanced-stage DLBCL who are PET-NEG at EOT and receive no RT have excellent outcomes. 18F-fluorodeoxyglucose-PET can reliably guide selective administration of consolidative RT, even in patients with initially bulky disease., (© 2021 by The American Society of Hematology.)

Cure rates for primary mediastinal large B-cell lymphoma (PMBCL) have improved with the integration of rituximab. However, the type of primary therapy and role of radiotherapy (RT) remains ill-defined. Herein, we evaluated the outcome of PMBCL primarily treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP) and the impact of an end-of-treatment (EOT) 18F-fluorodeoxyglucose positron emission tomography (PET) scan to guide consolidative RT. Patients ≥18 years of age with PMBCL treated with curative intent rituximab-chemotherapy were identified. Prior to 2005, patients were recommended to receive R-CHOP + RT (RT era). Beginning in 2005, EOT PET was used to guide RT and only those with a PET-positive scan received RT (PET era). In total, 159 patients were identified, 94% were treated with R-CHOP and 44% received RT (78% in RT era, 28% in PET era). The 5-year time to progression (TTP) and overall survival (OS) for the entire cohort were 80% and 89%, respectively, similar across treatment eras. Overall, 10% had refractory disease. In total, 113 patients had an EOT PET scan: 63% negative and 37% positive with a 5-year TTP of 90% vs 71% and 5-year OS of 97% vs 88%, respectively. For those with Deauville (D)-scored PET scans (n = 103), the 5-year TTP for PET-negative cases by Deauville criteria (D1-D3, DX) was 91%, with inferior outcomes for D5 vs D4 (5-year TTP 33% vs 87%, P = .0002). Outcomes for PMBCL treated with RCHOP are favorable and use of a PET-adapted approach reduces RT in the majority of patients. A small proportion have refractory disease and may benefit from an alternate treatment., (© 2020 by The American Society of Hematology.)

Mantle cell lymphoma (MCL) is an uncommon B-cell non-Hodgkin lymphoma (NHL) that is incurable with standard therapies. The genetic drivers of this cancer have not been firmly established, and the features that contribute to differences in clinical course remain limited. To extend our understanding of the biological pathways involved in this malignancy, we performed a large-scale genomic analysis of MCL using data from 51 exomes and 34 genomes alongside previously published exome cohorts. To confirm our findings, we resequenced the genes identified in the exome cohort in 191 MCL tumors, each having clinical follow-up data. We confirmed the prognostic association of TP53 and NOTCH1 mutations. Our sequencing revealed novel recurrent noncoding mutations surrounding a single exon of the HNRNPH1gene. In RNA-seq data from 103 of these cases, MCL tumors with these mutations had a distinct imbalance of HNRNPH1 isoforms. This altered splicing of HNRNPH1 was associated with inferior outcomes in MCL and showed a significant increase in protein expression by immunohistochemistry. We describe a functional role for these recurrent noncoding mutations in disrupting an autoregulatory feedback mechanism, thereby deregulating HNRNPH1 protein expression. Taken together, these data strongly imply a role for aberrant regulation of messenger RNA processing in MCL pathobiology., (© 2020 by The American Society of Hematology.)

Gray zone lymphoma (GZL), a B-cell lymphoma with features intermediate between large B-cell lymphoma (LBCL) and classic Hodgkin lymphoma (cHL), is a rare and poorly defined entity. Alongside GZL, a subset of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) has been described with polymorphic/GZL-like morphology (polymorphic-EBV-L). To fill the important gap in our understanding of the pathogenic process underlying these entities, we performed a gene expression study of a large international cohort of GZL and polymorphic-EBV-L, combined with cHL and primary mediastinal large B-cell lymphoma (PMBCL) cases. In an unsupervised principal component analysis, GZL cases presented with intermediate scores in a spectrum between cHL and PMBCL, whereas polymorphic-EBV-L clustered distinctly. The main biological pathways underlying the GZL spectrum were related to cell cycle, reflecting tumor cell content, and extracellular matrix signatures related to the cellular tumor microenvironment. Differential expression analysis and phenotypic characterization of the tumor microenvironment highlighted the predominance of regulatory macrophages in GZL compared with cHL and PMBCL. Two distinct subtypes of GZL were distinguishable that were phenotypically reminiscent of PMBCL and DLBCL, and we observed an association of PMBCL-type GZL with clinical presentation in the "thymic" anatomic niche. In summary, gene expression profiling (GEP) enabled us to add precision to the GZL spectrum, describe the biological distinction compared with polymorphic-EBV-L, and distinguish cases with and without thymic involvement as 2 subgroups of GZL, namely PMBCL-like and DLBCL-like GZL., (© 2020 by The American Society of Hematology.)

Diffuse large B-cell lymphoma (DLBCL) is the most common histologic subtype of non-Hodgkin lymphoma and is notorious for its clinical heterogeneity. Patient outcomes can be predicted by cell-of-origin (COO) classification, demonstrating that the underlying transcriptional signature of malignant B-cells informs biological behavior in the context of standard combination chemotherapy regimens. In the current study, we used mass cytometry (CyTOF) to examine tumor phenotypes at the protein level with single cell resolution in a collection of 27 diagnostic DLBCL biopsy specimens from treatment naïve patients. We found that malignant B-cells from each patient occupied unique regions in 37-dimensional phenotypic space with no apparent clustering of samples into discrete subtypes. Interestingly, variable MHC class II expression was found to be the greatest contributor to phenotypic diversity. Within individual tumors, a subset of cases showed multiple phenotypic subpopulations, and in one case, we were able to demonstrate direct correspondence between protein-level phenotypic subsets and DNA mutation-defined subclones. In summary, CyTOF analysis can resolve both intertumoral and intratumoral heterogeneity among primary samples and reveals that each case of DLBCL is unique and may be comprised of multiple, genetically distinct subclones. © 2019 International Society for Advancement of Cytometry., (© 2019 International Society for Advancement of Cytometry.)

Transmembrane protein 30A (TMEM30A) maintains the asymmetric distribution of phosphatidylserine, an integral component of the cell membrane and 'eat-me' signal recognized by macrophages. Integrative genomic and transcriptomic analysis of diffuse large B-cell lymphoma (DLBCL) from the British Columbia population-based registry uncovered recurrent biallelic TMEM30A loss-of-function mutations, which were associated with a favorable outcome and uniquely observed in DLBCL. Using TMEM30A-knockout systems, increased accumulation of chemotherapy drugs was observed in TMEM30A-knockout cell lines and TMEM30A-mutated primary cells, explaining the improved treatment outcome. Furthermore, we found increased tumor-associated macrophages and an enhanced effect of anti-CD47 blockade limiting tumor growth in TMEM30A-knockout models. By contrast, we show that TMEM30A loss-of-function increases B-cell signaling following antigen stimulation-a mechanism conferring selective advantage during B-cell lymphoma development. Our data highlight a multifaceted role for TMEM30A in B-cell lymphomagenesis, and characterize intrinsic and extrinsic vulnerabilities of cancer cells that can be therapeutically exploited.

Post-transplant lymphoproliferative disorder (PTLD) may arise after solid organ transplantation, and the most common subtype resembles diffuse large B cell lymphoma (DLBCL). In DLBCL-type PTLD, the anti-CD20 antibody rituximab (R) may be combined with chemotherapy (R-CHOP) or use a strategy (R-primary; similar to the PTLD-1 clinical trial) consisting of induction with four weekly doses of R-alone, without any chemotherapy or sequential R-CHOP follow-up. Here we report on a multicentre retrospective cohort of solid organ transplant patients with DLBCL-type PTLD that were treated with R. In 168 adults, two-year overall survival (OS) was 63·7% [95% CI (confidence interval) 56·6-71·7%]. No difference in OS was observed, whether patients were treated with R-CHOP versus the R-primary strategy. In the 109 patients treated with R-primary, multivariate analysis found that baseline IPI score and the response to R-induction predicted OS. Patients who responded to R-induction had durable remissions without the addition of chemotherapy. Conversely, of the 46 patients who had stable or progressive disease after R-induction (R-failure), those who received R-CHOP had an only marginally improved outcome, with a two-year OS of 45% (23·1-65·3%) vs. no R-CHOP at 32% (14·7-49·8%). In real-world patients, R-failure and high IPI scores predict a poor outcome in DLBCL-type PTLD., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

A novel prognostic score (IPS-3), comprised of only three of the seven IPS-7 indicators (age ≥45, stage IV, haemoglobin <105 g/l), was recently proposed as a simplified model for advanced-stage classic Hodgkin lymphoma (cHL). We aimed to validate this model in advanced-stage cHL patients treated with doxorubicin, bleomycin, vinblastine, dacarbazine (ABVD) in British Columbia. The estimated five-year freedom from progression (FFP) for scores of 0, 1, 2 and 3 were very similar to the original report at 84%, 76%, 72% and 68% respectively. The IPS-3 score is highly reproducible in this independent dataset and its simplicity makes it appealing for everyday clinical practice., (© 2019 British Society for Haematology and John Wiley & Sons Ltd.)

The objective of this study was to evaluate the distribution and prognostic impact of a broad range of molecular attributes in a large cohort of immunocompetent patients with primary central nervous system lymphoma (PCNSL) by using tissue microarray. Patients diagnosed with PCNSL were initially identified in the BC Cancer Lymphoid Cancer clinical and pathology databases. Tissue microarrays were constructed by using archival formalin-fixed paraffin-embedded diagnostic biopsy tissue. Immunohistochemistry and fluorescent in situ hybridization studies were performed. A total of 115 patients with PCNSL with diffuse large B-cell lymphoma (DLBCL) histology were identified. The majority of cases (≥75%) had a non-germinal center B-cell phenotype according to immunohistochemistry algorithms, but cell of origin did not affect progression-free or overall survival. MYC (40%), BCL2 (75%), and programmed death-ligand 1 (29%) protein expression were common, but their corresponding gene rearrangements were rare (≤1% each), suggesting that alternate mechanisms were driving expression. There were no dual rearrangements involving MYC and BCL2. Only 22% of cases had membranous expression of major histocompatibility complex class II, suggesting a mechanism for escape from immune surveillance. Epstein-Barr virus-encoded RNA was positive in 1 immunocompetent patient. BCL6 protein expression (77%) and BCL6 rearrangements (31%) were frequent; the latter was the only factor associated with a poor prognosis in the overall cohort and in the subgroup of 52 patients treated with high-dose methotrexate-based regimens. This large population-based study shows that prominent molecular features of PCNSL are unique and different from those of systemic DLBCL. These results may better inform drug development in PCNSL., (© 2019 by The American Society of Hematology.)

Despite widespread use of bendamustine and rituximab (BR) as frontline therapy for advanced-stage follicular lymphoma (FL), little is known about the risk of early progression or incidence of histological transformation. We performed a retrospective analysis of a population-based cohort of 296 patients with advanced-stage FL treated with frontline BR and maintenance rituximab. As previously demonstrated, outcomes with this regimen are excellent, with 2-year event-free survival estimated at 85% (95% confidence interval [95% CI], 80-89) and 2-year overall survival 92% (95% CI, 88-95). Progression of disease within 24 months (POD24) occurred in 13% of patients and was associated with a significantly inferior outcome with 2-year overall survival of 38% (95% CI, 20-55). The only significant risk factor for POD24 at baseline was elevated lactate dehydrogenase ( P < .001). Importantly, the majority of POD24 patients (76%) had transformed disease. Compared with a historical cohort treated with rituximab, cyclophosphamide, vincristine, and prednisone, event-free survival has improved and the risk of POD24 has decreased, but a higher proportion of patients with POD24 harbor transformation. The overall incidence of transformation appears unchanged. The presence of occult or early transformation is the main driver of POD24 in FL patients treated with frontline BR. Identification of biomarkers and improved management strategies for transformation will be crucial to improving outcomes., (© 2019 by The American Society of Hematology.)

Purpose: High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) has a poor outcome after standard chemoimmunotherapy. We sought to understand the biologic underpinnings of HGBL-DH/TH with BCL2 rearrangements (HGBL-DH/TH- BCL2) and diffuse large B-cell lymphoma (DLBCL) morphology through examination of gene expression., Patients and Methods: We analyzed RNA sequencing data from 157 de novo germinal center B-cell-like (GCB)-DLBCLs, including 25 with HGBL-DH/TH- BCL2, to define a gene expression signature that distinguishes HGBL-DH/TH- BCL2 from other GCB-DLBCLs. To assess the genetic, molecular, and phenotypic features associated with this signature, we analyzed targeted resequencing, whole-exome sequencing, RNA sequencing, and immunohistochemistry data., Results: We developed a 104-gene double-hit signature (DHITsig) that assigned 27% of GCB-DLBCLs to the DHITsig-positive group, with only one half harboring MYC and BCL2 rearrangements (HGBL-DH/TH- BCL2). DHITsig-positive patients had inferior outcomes after rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone immunochemotherapy compared with DHITsig-negative patients (5-year time to progression rate, 57% and 81%, respectively; P < .001), irrespective of HGBL-DH/TH- BCL2 status. The prognostic value of DHITsig was confirmed in an independent validation cohort. DHITsig-positive tumors are biologically characterized by a putative non-light zone germinal center cell of origin and a distinct mutational landscape that comprises genes associated with chromatin modification. A new NanoString assay (DLBCL90) recapitulated the prognostic significance and RNA sequencing assignments. Validating the association with HGBL-DH/TH- BCL2, 11 of 25 DHITsig-positive-transformed follicular lymphomas were classified as HGBL-DH/TH- BCL2 compared with zero of 50 in the DHITsig-negative group. Furthermore, the DHITsig was shared with the majority of B-cell lymphomas with high-grade morphology tested., Conclusion: We have defined a clinically and biologically distinct subgroup of tumors within GCB-DLBCL characterized by a gene expression signature of HGBL-DH/TH- BCL2. This knowledge has been translated into an assay applicable to routinely available biopsy samples, which enables exploration of its utility to guide patient management.