Super-enhancer hypermutation alters oncogene expression in B cell lymphoma.

Diffuse large B cell lymphoma (DLBCL) is the most common B cell non-Hodgkin lymphoma and remains incurable in around 40% of patients. Efforts to sequence the coding genome identified several genes and pathways that are altered in this disease, including potential therapeutic targets1-5. However, the non-coding genome of DLBCL remains largely unexplored. Here we show that active super-enhancers are highly and specifically hypermutated in 92% of samples from individuals with DLBCL, display signatures of activation-induced cytidine deaminase activity, and are linked to genes that encode B cell developmental regulators and oncogenes. As evidence of oncogenic relevance, we show that the hypermutated super-enhancers linked to the BCL6, BCL2 and CXCR4 proto-oncogenes prevent the binding and transcriptional downregulation of the corresponding target gene by transcriptional repressors, including BLIMP1 (targeting BCL6) and the steroid receptor NR3C1 (targeting BCL2 and CXCR4). Genetic correction of selected mutations restored repressor DNA binding, downregulated target gene expression and led to the counter-selection of cells containing corrected alleles, indicating an oncogenic dependency on the super-enhancer mutations. This pervasive super-enhancer mutational mechanism reveals a major set of genetic lesions deregulating gene expression, which expands the involvement of known oncogenes in DLBCL pathogenesis and identifies new deregulated gene targets of therapeutic relevance.

Unbalanced MYC break-apart FISH patterns indicate the presence of a MYC rearrangement in HGBCL-DH-BCL2.

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, where 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 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 mRNA 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.

Motive and opportunity: MYC rearrangements in high-grade B-cell lymphoma with MYC and BCL2 rearrangements (an LLMPP study).

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.

Genetic subdivisions of follicular lymphoma defined by distinct coding and noncoding mutation patterns.

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.

Molecular determinants of clinical outcomes in a real-world diffuse large B-cell lymphoma population.

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.

Genetic subgroups inform on pathobiology in adult and pediatric Burkitt lymphoma.

Burkitt lymphoma (BL) accounts for most pediatric non-Hodgkin lymphomas, being less common but significantly more lethal when diagnosed in adults. Much of the knowledge of the genetics of BL thus far has originated from the study of pediatric BL (pBL), leaving its relationship to adult BL (aBL) and other adult lymphomas not fully explored. We sought to more thoroughly identify the somatic changes that underlie lymphomagenesis in aBL and any molecular features that associate with clinical disparities within and between pBL and aBL. Through comprehensive whole-genome sequencing of 230 BL and 295 diffuse large B-cell lymphoma (DLBCL) tumors, we identified additional significantly mutated genes, including more genetic features that associate with tumor Epstein-Barr virus status, and unraveled new distinct subgroupings within BL and DLBCL with 3 predominantly comprising BLs: DGG-BL (DDX3X, GNA13, and GNAI2), IC-BL (ID3 and CCND3), and Q53-BL (quiet TP53). Each BL subgroup is characterized by combinations of common driver and noncoding mutations caused by aberrant somatic hypermutation. The largest subgroups of BL cases, IC-BL and DGG-BL, are further characterized by distinct biological and gene expression differences. IC-BL and DGG-BL and their prototypical genetic features (ID3 and TP53) had significant associations with patient outcomes that were different among aBL and pBL cohorts. These findings highlight shared pathogenesis between aBL and pBL, and establish genetic subtypes within BL that serve to delineate tumors with distinct molecular features, providing a new framework for epidemiologic, diagnostic, and therapeutic strategies.

Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing.

BACKGROUND: Somatic hypermutation (SHM) status of the immunoglobulin heavy variable (IGHV) gene plays a crucial role in determining the prognosis and treatment of patients with chronic lymphocytic leukemia (CLL). A common approach for determining SHM status is multiplex polymerase chain reaction and Sanger sequencing of the immunoglobin heavy locus; however, this technique is low throughput, is vulnerable to failure, and does not allow multiplexing with other diagnostic assays. METHODS: Here we designed and validated a DNA targeted capture approach to detect immunoglobulin heavy variable somatic hypermutation (IGHV SHM) status as a submodule of a larger next-generation sequencing (NGS) panel that also includes probes for ATM, BIRC3, CHD2, KLHL6, MYD88, NOTCH1, NOTCH2, POT1, SF3B1, TP53, and XPO1. The assay takes as input FASTQ files and outputs a report containing IGHV SHM status and V allele usage following European Research Initiative on CLL guidelines. RESULTS: We validated the approach on 35 CLL patient samples, 34 of which were characterized using Sanger sequencing. The NGS panel identified the IGHV SHM status of 34 of 35 CLL patients. We showed 100% sensitivity and specificity among the 33 CLL samples with both NGS and Sanger sequencing calls. Furthermore, we demonstrated that this panel can be combined with additional targeted capture panels to detect prognostically important CLL single nucleotide variants, insertions/deletions, and copy number variants (TP53 copy number loss). CONCLUSIONS: A targeted capture approach to IGHV SHM detection can be integrated into broader sequencing panels, allowing broad CLL prognostication in a single molecular assay.

A nu mouse model of diffuse large B-cell lymphoma in constitutional Atm loss.

Not available.

Characterization of DLBCL with a PMBL gene expression signature.

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.

The double-hit signature identifies double-hit diffuse large B-cell lymphoma with genetic events cryptic to FISH.

High-grade B-cell lymphomas with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/THs) include a group of diffuse large B-cell lymphomas (DLBCLs) with inferior outcomes after standard chemoimmunotherapy. We recently described a gene expression signature that identifies 27% of germinal center B-cell DLBCLs (GCB-DLBCLs) as having a double-hit-like expression pattern (DHITsig) and inferior outcomes; however, only half of these cases have both MYC and BCL2 translocations identifiable using standard breakapart fluorescence in situ hybridization (FISH). Here, 20 DHITsig+ GCB-DLBCLs apparently lacking MYC and/or BCL2 rearrangements underwent whole-genome sequencing. This revealed 6 tumors with MYC or BCL2 rearrangements that were cryptic to breakapart FISH. Copy-number analysis identified 3 tumors with MYC and 6 tumors with MIR17HG gains or amplifications, both of which may contribute to dysregulation of MYC and its downstream pathways. Focal deletions of the PVT1 promoter were observed exclusively among DHITsig+ tumors lacking MYC translocations; this may also contribute to MYC overexpression. These results highlight that FISH fails to identify all HGBL-DH/THs, while revealing a range of other genetic mechanisms potentially underlying MYC dysregulation in DHITsig+ DLBCL, suggesting that gene expression profiling is more sensitive for identifying the biology underlying poor outcomes in GCB-DLBCL.

Integrated single cell analysis reveals co-evolution of malignant B cells and tumor micro-environment in transformed follicular lymphoma.

Histological transformation of follicular lymphoma (FL) to aggressive forms is associated with poor outcome. Phenotypic consequences of this evolution and its impact on the tumor microenvironment (TME) remain unknown. We perform single-cell whole genome sequencing (scWGS) and transcriptome sequencing (scWTS) of 11 paired pre/post-transformation patient samples and scWTS of additional samples from patients without transformation. Our analysis reveals evolutionary dynamics of transformation at single-cell resolution, highlighting a shifting TME landscape, with an emerging immune-cell exhaustion signature, co-evolving with the shifting malignant B phenotype in a regulatory ecosystem. Integration of scWGS and scWTS identifies malignant cell pathways upregulated during clonal tumor evolution. Using multi-color immunofluorescence, we transfer these findings to a TME-based transformation biomarker, subsequently validated in two independent pretreatment cohorts. Taken together, our results provide a comprehensive view of the combined genomic and phenotypic evolution of malignant cells during transformation and shifting crosstalk between malignant cells and the TME.

STAT6 mutations enriched at diffuse large B-cell lymphoma relapse reshape the tumor microenvironment.

Diffuse large B-cell lymphoma (DLBCL) relapses in approximately 40% of patients following frontline therapy. We reported that STAT6D419 mutations are enriched in relapsed/refractory DLBCL (rrDLBCL) samples, suggesting that JAK/STAT signaling plays a role in therapeutic resistance. We hypothesized that STAT6D419 mutations can improve DLBCL cell survival by reprogramming the microenvironment to sustain STAT6 activation. Thus, we investigated the role of STAT6D419 mutations on DLBCL cell growth and its microenvironment. We found that phospho-STAT6D419N was retained in the nucleus longer than phospho-STAT6WT following IL-4 stimulation, and STAT6D419N recognized a more restricted DNA-consensus sequence than STAT6WT. Upon IL-4 induction, STAT6D419N expression led to a higher magnitude of gene expression changes, but in a more selective list of gene targets compared with STATWT. The most significantly expressed genes induced by STAT6D419N were those implicated in survival, proliferation, migration, and chemotaxis, in particular CCL17. This chemokine, also known as TARC, attracts helper T-cells to the tumor microenvironment, especially in Hodgkin's lymphoma. To this end, in DLBCL, phospho-STAT6+ rrDLBCL cells had a greater proportion of infiltrating CD4+ T-cells than phospho-STAT6- tumors. Our findings suggest that STAT6D419 mutations in DLBCL lead to cell autonomous changes, enhanced signaling, and altered composition of the tumor microenvironment.

Biological heterogeneity in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is heterogeneous both in clinical outcomes and the underlying disease biology. Over the last 2 decades, several different approaches for dissecting biological heterogeneity have emerged. Gene expression profiling (GEP) stratifies DLBCL into 3 broad groups (ABC, GCB, and DZsig/MHG), each with parallels to different normal mature B cell developmental states and prognostic implications. More recently, several different genomic approaches have been developed to categorize DLBCL based on the co-occurrence of tumor somatic mutations, identifying more granular biologically unified subgroups that complement GEP-based approaches. We review the molecular approaches and clinical evidence supporting the stratification of DLBCL patients based on tumor biology. By offering a platform for subtype-guided therapy, these divisions remain a promising avenue for improving patient outcomes, especially in subgroups with inferior outcomes with current standard-of-care therapy.

Exploiting the fibroblast growth factor receptor-1 vulnerability to therapeutically restrict the MYC-EZH2-CDKN1C axis-driven proliferation in Mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a lethal hematological malignancy with a median survival of 4 years. Its lethality is mainly attributed to a limited understanding of clinical tumor progression and resistance to current therapeutic regimes. Intrinsic, prolonged drug treatment and tumor-microenvironment (TME) facilitated factors impart pro-tumorigenic and drug-insensitivity properties to MCL cells. Hence, elucidating neoteric pharmacotherapeutic molecular targets involved in MCL progression utilizing a global "unified" analysis for improved disease prevention is an earnest need. Using integrated transcriptomic analyses in MCL patients, we identified a Fibroblast Growth Factor Receptor-1 (FGFR1), and analyses of MCL patient samples showed that high FGFR1 expression was associated with shorter overall survival in MCL patient cohorts. Functional studies using pharmacological intervention and loss of function identify a novel MYC-EZH2-CDKN1C axis-driven proliferation in MCL. Further, pharmacological targeting with erdafitinib, a selective small molecule targeting FGFRs, induced cell-cycle arrest and cell death in-vitro, inhibited tumor progression, and improved overall survival in-vivo. We performed extensive pre-clinical assessments in multiple in-vivo model systems to confirm the therapeutic potential of erdafitinib in MCL and demonstrated FGFR1 as a viable therapeutic target in MCL.

Relapse timing is associated with distinct evolutionary dynamics in DLBCL.

Diffuse large B-cell lymphoma (DLBCL) is cured in over 60% of patients, but outcomes are poor for patients with relapsed or refractory disease (rrDLBCL). Here, we performed whole genome/exome sequencing (WGS/WES) on tumors from 73 serially-biopsied patients with rrDLBCL. Based on the observation that outcomes to salvage therapy/autologous stem cell transplantation are related to time-to-relapse, we stratified patients into groups according to relapse timing to explore the relationship to genetic divergence and sensitivity to salvage immunochemotherapy. The degree of mutational divergence increased with time between biopsies, yet tumor pairs were mostly concordant for cell-of-origin, oncogene rearrangement status and genetics-based subgroup. In patients with highly divergent tumors, several genes acquired exclusive mutations independently in each tumor, which, along with concordance of genetics-based subgroups, suggests that the earliest mutations in a shared precursor cell constrain tumor evolution. These results suggest that late relapses commonly represent genetically distinct and chemotherapy-naïve disease.

Relapse Timing Is Associated With Distinct Evolutionary Dynamics in Diffuse Large B-Cell Lymphoma.

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.

Single-cell profiling reveals a memory B cell-like subtype of follicular lymphoma with increased transformation risk.

Follicular lymphoma (FL) is an indolent cancer of mature B-cells but with ongoing risk of transformation to more aggressive histology over time. Recurrent mutations associated with transformation have been identified; however, prognostic features that can be discerned at diagnosis could be clinically useful. We present here comprehensive profiling of both tumor and immune compartments in 155 diagnostic FL biopsies at single-cell resolution by mass cytometry. This revealed a diversity of phenotypes but included two recurrent patterns, one which closely resembles germinal center B-cells (GCB) and another which appears more related to memory B-cells (MB). GCB-type tumors are enriched for EZH2, TNFRSF14, and MEF2B mutations, while MB-type tumors contain increased follicular helper T-cells. MB-type and intratumoral phenotypic diversity are independently associated with increased risk of transformation, supporting biological relevance of these features. Notably, a reduced 26-marker panel retains sufficient information to allow phenotypic profiling of future cohorts by conventional flow cytometry.

Inhalation by design: dual pharmacology ß-2 agonists/M3 antagonists for the treatment of COPD.

This paper describes the successful design and development of dual pharmacology ß-2 agonists-M3 antagonists, for the treatment of chronic obstructive pulmonary disorder using the principles of 'inhalation by design'. A key feature of this work is the combination of balanced potency and pharmacodynamic duration with desirable pharmacokinetic and material properties, whilst keeping synthetic complexity to a minimum.