SUMMARY Diffuse large B cell lymphoma (DLBCL) is the most common form of blood cancer and is characterized by a striking degree of genetic and clinical heterogeneity. This heterogeneity poses a major barrier to understanding the genetic basis of the disease and its response to therapy. Here, we performed an integrative analysis of whole exome sequencing and transcriptome sequencing in a cohort of 1001 DLBCL patients to comprehensively define the landscape of 150 genetic drivers of the disease. We characterized the functional impact of these genes using an unbiased CRISPR screen of DLBCL cell lines to define oncogenes that promote cell growth. A prognostic model comprising these genetic alterations outperformed current established methods: cell of origin, the International Prognostic Index comprising clinical variables, and dual MYC and BCL2 expression. These results comprehensively define the genetic drivers and their functional roles in DLBCL to identify new therapeutic opportunities in the disease.

Peripheral T-cell lymphoma (PTCL) is a group of complex clinicopathological entities, often associated with an aggressive clinical course. Angioimmunoblastic T-cell lymphoma (AITL) and PTCL-not otherwise specified (PTCL-NOS) are the 2 most frequent categories, accounting for >50% of PTCLs. Gene expression profiling (GEP) defined molecular signatures for AITL and delineated biological and prognostic subgroups within PTCL-NOS (PTCL-GATA3 and PTCL-TBX21). Genomic copy number (CN) analysis and targeted sequencing of these molecular subgroups revealed unique CN abnormalities (CNAs) and oncogenic pathways, indicating distinct oncogenic evolution. PTCL-GATA3 exhibited greater genomic complexity that was characterized by frequent loss or mutation of tumor suppressor genes targeting the CDKN2A/B-TP53 axis and PTEN-PI3K pathways. Co-occurring gains/amplifications of STAT3 and MYC occurred in PTCL-GATA3. Several CNAs, in particular loss of CDKN2A, exhibited prognostic significance in PTCL-NOS as a single entity and in the PTCL-GATA3 subgroup. The PTCL-TBX21 subgroup had fewer CNAs, primarily targeting cytotoxic effector genes, and was enriched in mutations of genes regulating DNA methylation. CNAs affecting metabolic processes regulating RNA/protein degradation and T-cell receptor signaling were common in both subgroups. AITL showed lower genomic complexity compared with other PTCL entities, with frequent co-occurring gains of chromosome 5 (chr5) and chr21 that were significantly associated with IDH2R172 mutation. CN losses were enriched in genes regulating PI3K–AKT–mTOR signaling in cases without IDH2 mutation. Overall, we demonstrated that novel GEP-defined PTCL subgroups likely evolve by distinct genetic pathways and provided biological rationale for therapies that may be investigated in future clinical trials.

Hepatosplenic T cell lymphoma (HSTL) is a rare and lethal lymphoma; the genetic drivers of this disease are unknown. Through whole exome sequencing of 68 HSTLs, we define recurrently mutated driver genes and copy number alterations in the disease. Chromatin modifying genes including SETD2, INO80 and ARID1B were commonly mutated in HSTL, affecting 62% of cases. HSTLs manifest frequent mutations in STAT5B (31%), STAT3 (9%), and PIK3CD (9%) for which there currently exist potential targeted therapies. In addition, we noted less frequent events in EZH2, KRAS and TP53. SETD2 was the most frequently silenced gene in HSTL. We experimentally demonstrated that SETD2 acts as a tumor suppressor gene. In addition, we found that mutations in STAT5B and PIK3CD activate critical signaling pathways important to cell survival in HSTL. Our work thus defines the genetic landscape of HSTL and implicates novel gene mutations linked to HSTL pathogenesis and potential treatment targets.

Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of mature T-cell neoplasms with a poor prognosis. Recently, mutations in TET2 and other epigenetic modifiers as well as RHOA have been identified in these diseases, particularly in angioimmunoblastic T-cell lymphoma (AITL). CD28 is the major co-stimulatory receptor in T-cells which, upon binding ligand, induces sustained T-cell proliferation and cytokine production when combined with T-cell receptor stimulation. We have identified recurrent mutations in CD28 in PTCLs. Two residues – D124 and T195 – were recurrently mutated in 11.3% of cases of AITL and in one case of PTCL, not otherwise specified (PTCL-NOS). Surface plasmon resonance analysis of mutations at these residues with predicted differential partner interactions showed increased affinity for ligand CD86 (residue D124) and increased affinity for intracellular adaptor proteins GRB2 and GADS/GRAP2 (residue T195). Molecular modeling studies on each of these mutations suggested how these mutants result in increased affinities. We found increased transcription of the CD28-responsive genes CD226 and TNFA in cells expressing the T195P mutant in response to CD3 and CD86 co-stimulation and increased downstream activation of NF-κB by both D124V and T195P mutants, suggesting a potential therapeutic target in CD28-mutated PTCLs.

One-third of peripheral T-cell lymphomas are “not otherwise specified” (PTCL-NOS), but they have been subdivided into 2 subgroups based on gene expression profiling. Amador and colleagues generated an immunohistochemical algorithm that parallels the molecular separation of PTCL-NOS and provides useful prognostic information.

The adult high-grade B-cell lymphomas sharing molecular features with Burkitt lymphoma (BL) are highly aggressive lymphomas with poor clinical outcome. High-resolution structural and functional genomic analysis of adult Burkitt lymphoma (BL) and high-grade B-cell lymphoma with BL gene signature (adult-molecularly defined BL [mBL]) revealed the MYC-ARF-p53 axis as the primary deregulated pathway. Adult-mBL had either unique or more frequent genomic aberrations (del13q14, del17p, gain8q24, and gain18q21) compared with pediatric-mBL, but shared commonly mutated genes. Mutations in genes promoting the tonic B-cell receptor (BCR)→PI3K pathway ( and ) did not differ by age, whereas effectors of chronic BCR→NF-κB signaling were associated with adult-mBL. A subset of adult-mBL had translocation and mutation and elevated mRNA and protein expression, but had a mutation profile similar to mBL. These double-hit lymphomas may have arisen from a tumor precursor that acquired both and translocations and/or () mutation. Gain/amplification of and its paralogue loci was observed in 50% of adult-mBL. In vitro studies suggested's role in constitutive activation of BCR signaling and sensitivity to ibrutinib. Overall integrative analysis identified an interrelated gene network affected by copy number and mutation, leading to disruption of the p53 pathway and the BCR→PI3K or NF-κB activation, which can be further exploited in vivo by small-molecule inhibitors for effective therapy in adult-mBL.

B-cell non-Hodgkin’s lymphoma (B-NHL) encompasses multiple clinically and phenotypically distinct subtypes of malignancy with unique molecular etiologies. Common subtypes of B-NHL such as diffuse large B-cell lymphoma (DLBCL) have been comprehensively interrogated at the genomic level. But rarer subtypes such as mantle cell lymphoma (MCL) remain sparsely characterized. Furthermore, multiple B-NHL subtypes have thus far not been comprehensively compared using the same methodology to identify conserved or subtype-specific patterns of genomic alterations. Here, we employed a large targeted hybrid-capture sequencing approach encompassing 380 genes to interrogate the genomic landscapes of 685 B-NHL tumors at high depth; including DLBCL, MCL, follicular lymphoma (FL), and Burkitt lymphoma (BL). We identified conserved hallmarks of B-NHL that were deregulated in the majority of tumor from each subtype, including the frequent genetic deregulation of the ubiquitin proteasome system (UPS). In addition, we identified subtype-specific patterns of genetic alterations, including clusters of co-occurring mutations and DNA copy number alterations. The cumulative burden of mutations within a single cluster were more discriminatory of B-NHL subtypes than individual mutations, implicating likely patterns of genetic cooperation that contribute to disease etiology. We therefore provide the first cross-sectional analysis of mutations and DNA copy number alterations across major B-NHL subtypes and a framework of co-occurring genetic alterations that deregulate genetic hallmarks and likely cooperate in lymphomagenesis.

The activated B-cell (ABC-like) subtype of diffuse large B-cell lymphoma (DLBCL) is characterized by the chronic activation of signaling initiated by immunoglobulin-μ (IgM). By analyzing DNA copy profiles of 1,000 DLBCLs, we identified gains of 18q21.2 as the most frequent genetic alteration in ABC-like DLBCL. Using integrative analysis of matched gene expression profiling data we found that the TCF4 (E2–2) transcription factor gene is the target of these alterations. Over-expression of TCF4 led to its occupancy on immunoglobulin and MYC gene enhancers and increased their expression at the transcript and protein level. Inhibition of TCF4 activity with dominant-negative constructs was synthetically lethal to ABC-like DLBCL cell lines harboring TCF4 DNA copy gains, highlighting it as an attractive therapeutic target. Furthermore, the TCF4 gene is one of the top BRD4-regulated genes in DLBCL and a BET proteolysis-targeting chimera (PROTAC) extinguished TCF4, MYC and IgM expression and killed ABC-like DLBCL cells in vitro and in vivo. This highlights a novel genetic mechanism for promoting immunoglobulin signaling in ABC-like DLBCL and provides a functional rationale for the use of BET inhibitors in this disease.