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Newly Identified Mechanisms in B-Cell Non-Hodgkin Lymphomas Uncovered by Next-Generation Sequencing

Seminars in Hematology, 4, 50, pages 303 - 313

Application of molecular techniques to study the genetics and gene expression alterations in non-Hodgkin lymphomas (NHLs) has revealed examples of common genetic events shared across individual disease types as well as extensive heterogeneity within specific diseases. For example, diffuse large B-cell lymphoma (DLBCL) can be effectively divided into two broad molecular subgroups in which genetic changes unique to each subgroup continue to be further determined. The broad availability of next-generation sequencing (NGS) now affords the ability to fully characterize the genetics of individual tumor types and application of this to some common NHLs has provided a wealth of new information. NGS-based research in NHL has unraveled a complex set of genetic alterations that underlie each of the DLBCL molecular subgroups. For example, these and studies of other NHLs have indicated a variety of mechanisms by which nuclear factor- κB (NF-κB) is deregulated in mature B-cell neoplasms and have also found extensive common features between germinal center B-cell–like DLBCL and other germinal center–derived lymphomas. Overall, NGS has identified new players in each of the studied diseases and provided promising new therapeutic targets for NHL.

Non-Hodgkin lymphomas (NHLs) are cancers derived from lymphoid or immune-related cells including B cells, T cells, or natural killer cells at various stages of differentiation that collectively comprise more than 30 clinicopathologic entities. Historically, approaches towards discovering the genetic and molecular features of cancer were limited to either measurement of transcriptional output using gene expression profiling (GEP) or large-scale detection of unbalanced gains and losses of genetic material, including aneuploidies, known collectively as copy number alterations (CNAs). Some of the common and aggressive NHL types have been extensively studied using these and other, more targeted, molecular techniques such as candidate gene resequencing. Such research has lead to methods for improved molecular diagnoses of select NHL types1 and 2 and has yielded a wealth of knowledge of core signaling pathways that are deregulated or suppressed during lymphomagenesis. 3

New “next-generation” sequencing (NGS) technologies have emerged over the past 8 years and have afforded an opportunity to study genetic alterations present in cancer at an unprecedented resolution. RNA-seq, an early NGS application, 4 involves the sequencing of short fragments of cDNAs that collectively represent the entire complement of transcripts present in a tissue sample. Since the number of fragments sequenced is proportional to the number of copies of a given mRNA species, this technique yields gene expression information with improved dynamic range and breadth in comparison to array-based profiling methods. 5 As sequence information is simultaneously determined for each expressed transcript, this methodology has the potential to capture splicing events and has been demonstrated to be suitable for identifying genes involved in fusion transcripts and those affected by recurrent somatic point mutations.6 and 7

Owing to the steady decrease in sequencing cost and the concomitant increase in throughput of NGS, more global mutational surveys such as whole-genome sequencing (WGS) and exome sequencing have proliferated. Currently, the most widely adopted NGS platform is the HiSeq 2000 offered by Illumina (San Diego, CA), which can routinely produce paired reads of between 75 and 150 base pairs from the ends of approximately 1.5 x 108 DNA fragments from a single lane. In practice, one can attain 30-fold redundant coverage of the human genome by sequencing a sample using only three of the 16 lanes available on the instrument. Such WGS experiments offer the potential to uncover the full complement of somatic single-nucleotide variants (SNVs), insertions/deletions (indels), structural rearrangements, and copy number alterations (CNAs) across the entire genome of a tumor. Exome sequencing involves a process that captures only the DNA fragments corresponding to exons and is restricted to detecting SNVs and small indels that affect the exons effectively targeted by the design of the exome kit, namely, what fraction of known exons and more recently regulatory regions and non-coding regulator RNA genes are also covered. Though gene expression information is not captured by either of these methods, they facilitate less biased assessment of the mutations across all genes regardless of their expression level. To facilitate recognition of the numerous rare “private” mutations present in individuals, both tumor DNA and a matched constitutional (germ-line) sample are typically sequenced in concert. By sequencing many tumors of the same diagnosis in this way and applying rigorous statistical techniques to identify significantly mutated genes, WGS and exome sequencing studies have the potential to reveal all significant targets of somatic point mutation in a given cancer type.8 and 9

In this review, we provide an overview of the large studies that have applied NGS to identify the spectrum of mutations in NHL and discuss some novel discoveries stemming from these. As will be discussed in detail, many of the common NHLs have now been rigorously analyzed by RNA-seq,6, 7, 10, and 11 exome,10, 12, and 13 and genome10, 13, and 14 sequencing and these studies have collectively uncovered a new layer of complexity in these diseases and have added an unprecedented number of new mutation targets (genes) likely to be relevant to individual cancers. We also highlight some novel features of individual NHL types and some pan-lymphoma trends that have emerged as a result of these efforts and discuss the possible impact these observations may have on clinical management of these diseases.

Genetic Heterogeneity Within and Among Clinical Entities

Historically, some of the individual NHL types have been considered, at the genetic level, to be homogeneous whereas other diagnoses harbor a heterogeneous assortment of genetic alterations, a feature that lends itself to subdivision into molecular subgroups. For example, each of mantle cell lymphoma (MCL), Burkitt lymphoma (BL), and follicular lymphoma (FL) have long been characterized by the presence of prototypic translocations but, in contrast, no such unifying genetic alteration has been identified in diffuse large B-cell lymphoma (DLBCL). Individual DLBCL cases commonly share cytogenetic features with other NHL types such as t(3;14)(q27;q32), t(14;18)(q32;q21), and t(8;14)(q24;q32), but individually these are found in approximately 35%, 15%, and 10% of samples, respectively ( Figure 1 ). A seminal transformative discovery made using GEP was the observation that some of the heterogeneity among DLBCL could be robustly captured at the gene expression level, a finding that ultimately spurred the separation of DLBCL into at least two molecular subgroups. 3 The genes expressed more abundantly in the activated B-cell (ABC) subgroup of DLBCL were found to be enriched for targets of NF-κB, suggesting that this signaling pathway is constitutively activated in ABC DLBCL. Subsequent molecular profiling of DLBCL using a combination of GEP and high-resolution DNA copy number arrays demonstrated that certain recurrent CNAs differ in prevalence between ABC and GCB cases. These included the identification of more GCB-specific alterations beyond such classic lesions as t(14;18), including loss of the PTEN tumor-suppressor and amplification of the oncogenic microRNA cluster miR-17~92, both of which contribute to enhanced Akt/mTOR activity and block cell death. 15 These observations strengthened the notion that GCB and ABC arise from divergent pathways and set the stage for a plethora of subsequent functional 16 and genetic 17 studies that have extensively catalogued numerous additional molecular and genetic differences between the subgroups (reviewed in detail elsewhere18 and 19). Despite advancement of our understanding of the molecular underpinnings of DLBCL, prior to the application of NGS to these diseases, only a subset of ABC DLBCL could be explained by the known genetic aberrations and the genetic causal mutations that underlie GCB cases and the numerous other NHLs remained poorly understood.

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Figure 1 Comparison of incidence of novel mutation targets identified by NGS to some classical lesions. The incidence of mutations affecting some of the genes identified by NGS is shown for various NHL types with individual diseases roughly ordered by the B-cell differentiation stage from which each is thought to derive. Multiple genes mutated more commonly in GCB DLBCL are also found mutated in FL such as EZH2 and MEF2B. FL and MCL have not yet been globally profiled for mutations so whether these diseases also regularly harbor mutations in other GCB-related genes such as GNA13 and SGK1 is not yet known. In addition to the NOTCH mutations discussed in the text, targeted sequencing in MCL has identified recurrent inactivating mutations in UBR5, 54 which encodes an E3 ubiquitin ligase gene.

Oncogenic Pathways Shared Between ABC DLBCL and Mature B-Cell Neoplasms

Constitutive activity of the NF-κB pathway is a key signature that is commonly observed in ABC DLBCL and is also known to be an important pathway in other mature B-cell cancers. In the past decade, progress has been made in uncovering the multitude of genetic mechanisms by which NF-κB activity can be altered in ABC DLBCL. Prior to the availability of NGS, elegantly designed synthetic lethality RNA interference screens enabled the detection of genes required for survival in individual ABC cell lines. 16 Some of the genes identified by this approach were already reported to be mutated or deregulated in some NHLs. Other candidate genes identified as necessary for mediating survival in these assays, for example CARD11 20 and CD79B, 21 were subsequently sequenced and found to harbor oncogenic mutations in primary samples and cell lines.

RNA-seq was the first NGS approach used to analyze ABC DLBCL for somatic mutations in a broad and unbiased fashion. The analysis identified MYD88 as yet another gene with apparent dominant-acting mutations. By screening this gene for mutations in a large cohort using classical Sanger sequencing, MYD88 mutations were found to be largely absent from GCB cases and other NHL types. 7 Overall MYD88 mutations were present in 39% of all ABC-type tumors, positioning it among the most frequently altered genes in this disease. The most commonly observed (and most potent) mutation identified in DLBCL was replacement of leucine with proline at codon 265 (L265P). This mutant form of MYD88 was shown to complex with IRAK1 and activate NF-κB via an alternative mechanism while also activating JAK-STAT3 signaling, 7 further supporting the model of ABC DLBCL being largely driven by constitutive NF-κB activity.

Multiple myeloma (MM) and lymphoplasmacytic lymphoma (LPL) are cancers that derive from mature (post-germinal center) B cells. It has long been appreciated that the RAS and NF-κB pathways are both active in MM cells. Interestingly, WGS analysis of 38 MM patients for point mutations and structural alterations did not uncover any highly recurrent targets of mutation beyond those already known. 22 Instead, the authors of this study highlighted the numerous infrequent mutations distributed across genes involved in NF-κB signaling, as well as multiple genes known to be involved in post-translational modification of histone proteins (histone modifiers). It is unclear whether this finding indicates that MM is a heterogeneous disease driven by numerous infrequent mutations or, alternatively, that the most significant genetic alterations that contribute to MM, namely, the translocations that deregulate various oncogenes, had already been characterized.

Chronic lymphocytic leukemia (CLL) can derive from post-germinal center B cells as well as antigen naïve B cells, resulting in IGHV-mutated and unmutated varieties. A small study analyzed four CLL cases by exome sequencing and identified only 45 genes affected by non-silent SNVs. 22a By screening additional cases for mutations in these genes, four recurrently mutated genes were identified including MYD88, XPO1, KLHL6, and NOTCH1 (discussed in detail in a subsequent section). The same MYD88 mutation L265P, commonly observed in ABC DLBCL was observed. This mutation was reported here to be more common in the post-germinal center (IGHV-mutated) cases, further supporting that MYD88 mutations are a feature of post-germinal center neoplasms. A larger follow-up study of a total of 105 CLL cases identified additional genes not captured in this preliminary screen. 23 One such gene was SF3B1, which encodes a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP). In contrast to MYD88, this gene was more commonly mutated in IGHV-unmutated (pre-germinal center) cases ( Figure 1 ).

In stark contrast to the heterogeneity observed in MM and CLL, another team applied WGS to 30 LPL patients and identified MYD88 L265P as a single recurrent mutation present in virtually all cases analyzed, 24 indicating that deregulation of NF-κB may also be a feature of LPL. Beyond MYD88, multiple examples of mutations affecting MLL2 and ARID1A were reported in this study. Both of these encode proteins that modulate chromatin structure could have an impact on the epigenome of LPL. The observation of mutations in such genes will be reviewed in detail in a subsequent section.

Germinal Center B-Cell Lymphomas

Global screening of DLBCL and other NHLs using NGS has helped to strengthen the widely held view that lymphomas deriving from the germinal center have common genetic underpinnings and has extended our understanding of this group of NHLs (namely, GCB DLBCL, FL, and BL). Many of the initial NGS-based studies of NHL established a significant overlap in the genes mutated in both FL and DLBCL; in particular, they have reported a consistent enrichment for mutation targets shared between FL and GCB cases. The first such study included a single FL patient and a modest number of DLBCL but nonetheless identified a recurrent mutation affecting a single codon (641) in the EZH2 gene ( Table 1 ). 6 Similar to t(14;18) and TP53 mutations, this mutation was more commonly observed in GCB DLBCL and also present in FLs. A follow-up study including an expanded number of DLBCL as well as other NHL types reported a larger list of GCB-restricted mutation targets ( Table 1 ). These included those previously reported, BCL2, EZH2, and TP53, as well as three genes not previously identified as mutation targets in DLBCL: TNFRSF14, MEF2B, SGK1, and GNA13. 10 None of these four genes had been implicated in the known NHL pathways and only one, TNFRSF14, had previously been identified as a candidate tumor-suppressor gene. 25

Table 1 NGS-Based Lymphoma Studies

Diseases Cohort Size Sequencing Methods Key Findings Reference
DLBCL, FL 1 FL, 38 DLBCL Genome, RNA-seq EZH2 hot spot Morin et al, 2010 6
MM 36 Genome Histone modifiers, NF-kB Chapman et al, 2011 22
PMBCL and HL 2 cell lines RNA-seq CIITA fusions Steidl et al, 2011 42
DLBCL 4 cell lines RNA-seq MYD88 hot spots Ngo et al. 2011 7
DLBCL 6 Exome Loss of CREBBP, EP300 Pasqualucci et al, 2011 26
CLL 4 Exome, Genome NOTCH1, MYD88 Puente et al, 2011 22a
DLBCL, FL, other NHLs 14 WGS, 129 total Exome, Genome, RNA-seq MLL2, MEF2B, GNA13, SGK1 Morin et al, 2011 10
DLBCL, FL 6 Exome * MLL2 mutation Pasqualucci et al, 2011b 27
MCL 18 RNA-seq NOTCH1 mutation Kridel et al, 2011 11
CLL 105 Exome SF3B1 mutation Quesada et al, 2011 23
LPL 30 Genome MYD88 hot spot Treon et al, 2012 24
DLBCL 55 Exome   Lohr et al, 2012 12
DLBCL 96 RNA-seq TBL1RXR1-TP63 Scott et al, 2012 37
BL 28 BL, 13 CL RNA-seq ID3, TCF3, CCND3 Schmitz et al, 2012 36
BL 4 BL WGS, RNA-seq, exome ID3 Richter et al, 2012 35
BL 14 adult, 13 ped, 24 NA, 8 CL Exome ID3 Love et al, 2012 13
SMZL 6 WGS NOTCH2 Kiel et al, 2012 51
SMZL 8 Exome * NOTCH2 Rossi et al, 2012 52
DLBCL 73 cases and 21 cell lines Exome * PIK3CD, MTOR mutations Zhang et al, 2013 14
DLBCL 40 cases and 13 cell lines WGS 41 novel mutation targets Morin et al, 2013 30

lowast Gene described in study was not found mutated in initial global screen in these studies. For MLL2, probes were not present in the exome kits used in these studies and thus the gene was not covered. MLL2 was sequenced in Pasqualucci et al for completeness. Similarly, for NOTCH2, this gene was not identified by exome sequencing but re-sequenced in the extension cohort owing to presence of mutations in NOTCH1.

A separate group employed a combination of exome sequencing, array-based copy number analysis and targeted resequencing to screen DLBCL for potential tumor-suppressor genes, which are expected to be characterized by recurrent genomic loss and mutational inactivation. This approach facilitated the identification of two additional genes as recurrent targets of SNVs and CNVs: and EP300. 26 It was reported that CREBBP mutations were enriched in GCB DLBCL as well as FL CREBBP and this gene was posited as one of the most commonly mutated genes in both diseases. Because SNVs in CREBBP and EP300 have also been observed in a significant proportion of ABC cases10 and 26 and these SNVs were not found to be GCB-enriched in other studies,10 and 14 it remains unclear whether these mutations are specific to germinal center neoplasms ( Figure 1 ). It is conceivable that the inclusion of copy number data and the use of DLBCL-derived cell lines for estimating the prevalence of mutations affecting these genes could have had an impact on this analysis; further work is needed to resolve this issue.

With additional NGS sequencing studies applied to DLBCL12 and 27 ( Table 1 ), the genes commonly mutated in DLBCL and those more prevalent in GCB lymphomas continue to emerge. A recent large exome-based study of 73 primary DLBCL confirmed the restriction of EZH2, BCL2, and GNA13 mutations to GCB cases and identified an additional GCB-restricted mutation target (PCDHGA2), as well as multiple genes apparently more commonly mutated in ABC cases. 14 As noted by the authors, mutations in many of the genes reported in this study, including PCDHGA2, were not significantly mutated in other DLBCL cohorts such as that screened by Morin et al using RNA-seq 10 or the exome-based screens conducted Pasqualucci et al 27 or Lohr et al. 12 An important caveat inherent in this study was that constitutional DNA was not sequenced in parallel for the majority and thus some of the events reported may represent rare/private germline mutations rather than acquired somatic mutations. Nonetheless, the large number of genes reported as significantly mutated in this study (n = 322) is striking, underscoring the need for larger surveys.

In another recent effort, the whole genomes of tumors and matched constitutional DNA from 40 DLBCL patients were sequenced. These data were first used to globally identify genes subject to aberrant somatic hypermutation (aSHM), a process previously known to have an impact on a small number of genes in DLBCL. 28 In addition to confirming 12 known aSHM targets, this analysis uncovered a suite of new aSHM targets (36 in total). Interestingly, the authors provided evidence that some of these aSHM targets are more commonly mutated in either ABC or GCB cases such as PIM1 and S1PR2, respectively, 29 further adding to the list of candidate subgroup-restricted mutation targets. Subsequent in-depth analysis of these genomes revealed many novel somatic mutations including complex structural alterations and small/focal deletions not detectable by previous study designs. This analysis also uncovered 41 genes significantly affected by SNVs that had not previously been reported as associated with DLBCL. The allelic ratios of point mutations derived from the genome sequence data were also used to approximate the order in which individual mutation events arose in individual tumors. This analysis suggested that well-characterized driver mutations such as amplification of BCL2 and point mutations in TP53, CARD11, MYD88 and CD79B can arise both early and late during lymphoma progression. 31

Despite substantial understanding of the molecular functions of some newly identified GCB-restricted genes, the underlying altered mechanisms that contribute to lymphomagenesis remains speculative. Constitutive AKT/mTOR activity is known to be a feature of GCB DLBCL and it is appealing to consider that some of these genes may impact activity of these pathways. SGK1 (encoded by SGK1) is a kinase with functional overlap with AKT, but the variants observed in this gene appear to result from aSHM and often include loss of function mutations.10 and 30 As increased (not decreased) AKT activity is known to drive survival in DLBCL and other NHLs, loss of SGK1 activity would be inconsistent with a direct functional overlap between SGK1 and AKT. FOXO1 is another gene that was identified as commonly mutated in FL 10 and DLBCL by NGS. 31 Both AKT and SGK1 are responsible for regulating subcellular localization and turnover of FOXO1 and each protein is responsible for phosphorylating separate residues on the protein. Considering that the FOXO1 mutations in DLBCL have an impact on the subcellular localization of FOXO1 protein, 31 it is feasible that loss of SGK1 activity could similarly affect the ability of the cell to regulate FOXO1 localization and thus transcriptional activity.

It was originally observed that GNA13 is mutated in up to 25% of GCB patients in one study 10 and the high prevalence of GNA13 mutations GCB DLBCLs was recently confirmed; however, the effects of these mutations have not yet been explained. 30 Similar to SGK1, the pattern of mutations observed in GNA13 in GCB DLBCL and FL indicate it likely acts as a tumor-suppressor in these diseases. 10 GNA13 encodes Gα13, a G-protein that couples with sphingosine-1-phospate (S1P) receptors and regulates Rho-mediated motility. 32 In germinal center B cells, Gα13 couples most frequently with S1P2 (encoded by S1PR2), a gene previously identified as mutated in DLBCL 33 and, as discussed above, its mutation is potentially GCB-restricted. 29 Further, GNAI2, a gene that encodes another Gα protein, which acts upstream of Rho signaling, was also recently found to be mutated in GCB DLBCLs. 30 In normal B cells, signaling via these proteins in response to S1P gradients serves to confine cells to the germinal center microenvironment by regulating motility via RhoA. 32 Notably, beyond altering the motility of B cells, the increase in Rho signaling induced by S1P2/Gα13 can lead to increased PTEN activity and, as a result, potent AKT inhibition. 34 It is plausible to hypothesize that mutations in these three genes could result in loss of confinement to the germinal center niche as well as possibly contributing to the deregulation of AKT/mTOR known to be a feature of GCB DLBCL.

BL is one of several NHL subtypes that derive from B cells in the germinal center. Though more prevalent in children, BL also affects a significant number of adults. It has long been appreciated that BL tumors, by definition, harbor a translocation that results in deregulated expression of MYC, but other than this event, few drivers of the disease had been characterized. Recent high-resolution (WGS, exome sequencing, and RNA-seq) profiling of BL cohorts by three separate groups revealed a suite of mutation targets, many of which were among those identified in DLBCL.13 and 35 Perhaps not surprisingly, certain genes with mutations restricted to GCB DLBCL and FL were also mutated in BL, whereas other mutations common in GCB DLBCL were notably absent. Unfortunately, the mutation status for some of the GCB-restricted genes was inconsistent between the studies ( Figure 1 ). For example, EZH2 and CREBBP mutations were found in multiple cases by Love et al 13 but were not reported by Schmitz et al, 36 possibly reflecting different proportions of adult BL cases included in the two studies. However, the lack of SGK1 mutations and presence of GNA13 mutations in BL was a consistent observation in both studies ( Figure 1 ). Interestingly, one group also noted recurrent mutations in RHOA among the index BL cases sequenced. 35 Mutations in RHOA have also been noted in a few DLBCL cases. 30 Although this observation has not been confirmed in a larger cohort in either disease, it provides further evidence that RhoA signalling (and perhaps Rho-mediated motility) is a feature of GCB NHLs.

Newly Identified Oncogenic Mutations in BL

The three large NGS studies of BL have uncovered multiple common features between BL and the GCB DLBCL, likely reflecting a common cell of origin. This had previously been expected, but it was also appreciated that at least some genetic alterations more common to ABC DLBCL could occur in BL. Specifically, loss of CDKN2A, which is common in ABC DLBCL, is also a known feature of BL. In addition to this, a single gene (CCND3) that was recently discovered more commonly mutated in ABC DLBCL was also mutated in BL. 36 Interestingly, these mutations were restricted to sporadic and human immunodeficiency virus (HIV)-associated BL cases but rare within the Epstein-Barr virus (EBV)-associated (endemic) BLs. The CCND3 mutations included multiple non-synonymous SNVs in a highly conserved region, as well as frameshift mutations removing the C-terminus of the protein. These mutations were shown to produce a form of the protein with an extended half-life. CCND3 encodes cyclin D3, a protein that complexes with CDK6 to regulate the G1–S transition in B cells. This study demonstrated that CCND3 mutations contribute to increased Cyclin D3 half-life and thus could drive proliferation in BLs.

Beyond mutations shared with DLBCL, additional genes were found mutated solely in BL with two examples that have been explored in detail, namely, the transcription factor TCF3 and its negative regulator ID3.13, 35, and 36 The variants observed in ID3 largely comprised nonsense and frameshift mutations, as well as a collection of missense SNVs in the region encoding the helix-loop-helix domain of the protein. 35 The ID3 mutations block its ability to negatively regulate TCF3. Interestingly, the TCF3 mutations clustered at residues that mediate the interaction between TCF3 and ID3 and these were also found to block the ability of these to proteins to dimerize, thereby relieving TCF3 of ID3-mediated inhibition. 36 GEP was applied in an attempt to uncover the genes with expression altered by deregulated TCF3 activity that results from the mutations. This identified ID3 as a transcriptional target of TCF3, as well as multiple genes important to the biology of germinal center B cells.

New Recurrent Gene Fusions

Genomic rearrangements resulting from double-strand breaks can reposition important genes such as oncogenes under the control of regulatory sequences belonging to genes that are often highly expressed, and thus deregulate expression in that cell. The expression of BCL2, BCL6, and other oncogenes is commonly maintained by such events in FLs and DLBCL. In some cases, genomic rearrangements can also generate a fusion transcript, which may be translated into a protein with a biologically distinct function. Beyond measuring the total transcriptional output of a gene, appropriate analysis of RNA-Seq data can expose the presence of such fusion events. To date, such analysis has enabled the detection of two novel genes that are involved in such fusion events in NHLs: CIITA and TP63 ( Table 1 ). The likely relevance of CIITA fusions, which have been observed in primary mediastinal B-cell lymphoma (PBMCL) and classical HL (cHL), is discussed in a subsequent section.

The second novel fusion protein identified in B-cell lymphomas involves TP63 (tumor protein p63) and TBL1XR1 (transducing (beta)-like 1 X-linked receptor 1) and was first identified in DLBCL and FL ( Figure 2 ). 37 Though recurrent, this event is rare in DLBCL (~2%) and possibly exclusive to GCB cases and has also been detected in FL. Despite the GCB predominance, a trend was noted towards more clinically aggressive disease in that three of the four DLCBL patients harboring this event had primary refractory disease. This fusion was later discovered to be more prevalent in peripheral T-cell lymphoma (PTCL), with 5.8% of cases in an extension cohort showing evidence for rearrangements involving TP63. 38 PTCL patients harboring this fusion also demonstrated poor survival, further suggesting that this fusion event results in more aggressive lymphomas.

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Figure 2 Recurrent TBL1XR1-TP63 fusion detected in DLBCL and FL. (A) Fusions between TBL1XR1 and TP63 were identified by RNA-seq analysis of DLBCL and FL. Mapping of short reads to the transcriptome and genome identified consistent pairing of reads between exons 6 and 7 of TBL1XR1 and exon 4 of TP63. (B) The fusion event can result from a 12.8 megabase inversion on chromosome 3.

Although the presence of a TBL1XR1-TP63 fusion protein has been detected by Western blot, it has not yet been determined whether this protein has a neomorphic function (ie, novel or enhanced enzymatic activity) or if the oncogenic effect of this event results from the disruption of one or both of the involved genes. 38 As the TP63 partner was confirmed to be TBLXR1 in only seven of the 11 cases with rearrangements detected by fluorescence in situ hybridization (FISH) in PTCL, this suggest that other TP63 fusions or disruptions of this gene may also exist. However, it has been noted that all TP63 rearrangements include exons 4 onward and thus contain the same domain structure as the ΔNp63 isoform of TP53, which lacks a fully functional transactivation domain. Importantly, this isoform of TP63 has a dominant-negative effect over the function of other p53 family members37 and 38. More recently, deletions removing portions of the TP63 gene but leaving transcription intact have been reported in DLBCL. 30 . Taken together, these observations suggest that these various mutations affecting TP63 mimic an isoform that can antagonize the function of p53.

Microenvironment Interaction and Immune Evasion

Early application of gene expression profiling to various lymphomas revealed evidence for an impact of the presence of non-malignant cells in the tumor microenvironment on patient outcome.39, 40, and 41 Multiple NGS-based studies have uncovered specific genetic alterations that point to mechanisms by which the interactions between tumor cells and their microenvironment may be altered. This began with the observation of recurrent rearrangements involving CIITA gene and concomitant deregulation of CD274 and CD273 (PDL1 and PDL2, respectively) by fusion or amplification in both PMBCL and cHL. 42 The rearrangements affecting CIITA result in reduced expression of major histocompatibility complex (MHC) class II and thus diminished recognition (and thus infiltration) by cytotoxic T cells. As these fusions concomitantly induced the expression level of PD-1 ligands (PDL1 and PDL2), PMBCL cells produce an imbalance between T-cell receptor and costimulatory signaling, which, in turn, results in T-cell anergy, apoptosis, and skewed differentiation toward regulatory T cells. 43

More recently, an analogous method of immune escape has been observed in DLBCL. Using RNA-seq and exome sequencing, loss-of-function mutations in B2M and CD58 were both found to be common in DLBCL.10 and 27 Subsequent analysis of each locus indicated both regions are also affected by recurrent copy number loss in DLBCL. 44 B2M encodes β2-microglobulin (B2M), which, together with the human leukocyte antigen (HLA) heavy chain, forms MHC class I. Analysis of DLBCL biopsies using immunohistochemistry demonstrated that the majority of samples are negative for B2M protein and these corresponded to loss of MHC class I expression. These included samples with mono- or bi-allelic mutations, as well as samples with seemingly wild-type B2M. As the MHC class I complex is responsible for presenting peptides to cytotoxic T cells, loss of this complex effectively enables DLBCL to evade immune surveillance. Similar to B2M, mutations in CD58 were typically accompanied by loss of protein expression or mis-localization of the protein in primary tumor samples and cell lines. CD58 protein is a ligand for the CD2 receptor on both T and natural killer (NK) cells. Hence, loss of CD58 expression is expected to reduce the recognition of DLBCL by these immune cells thereby reducing NK-mediated cytolysis. These discoveries demonstrate that mutations or gene expression alterations that allow tumor cells to evade or modulate immune cell interactions are likely selected for during lymphomagenesis and are likely to be a general feature of PMBCL and DLBCL.

Mutations Impacting Regulators of the Epigenome

Beginning with the observation of EZH2 mutations, 10 a significant trend among the mutations more common in GCB lymphomas is the predominance of genes directly involved in regulating the post-translational modification state of histone proteins. 45 Each of EZH2, CREBBP, and EP300 is responsible for methylating or acetylating histone lysine residues. The sole target of EZH2, the catalytic component of the polycomb repressive complex 2 (PRC2), is K27 on histone H3 (H3K27). Among other residues on H3 and other histones, CREBBP/EP300 target this residue for acetylation. The common EZH2 mutations (affecting Y641) were ultimately shown to enhance overall methyltransferase activity on H3K27. By targeting the same residue for a modification with an opposing impact on gene expression, the activity of CREBBP/EP300 can be considered antagonistic to that of EZH2. Given that the mutations of these acetyltransferases are clearly loss-of-function ( Figure 3 ), it is conceivable that the oncogenic effect of these mutations may converge to alter the expression of some of the same genes. MEF2B was also found to be a target of recurrent mutation in GCB DLBCL and FL. Though not itself a histone modifier, MEF2B dimers recognize MEF2-box sites on the DNA and can recruit both of these histone acetyltransferases, as well as numerous histone deacetylases to specific regions. Interestingly, there is some evidence that mutant MEF2B may deregulate the expression of BCL6, 46 Thus, MEF2B mutations provide another potential mechanism by which EZH2- and CREBBP/EP300-mediated histone modification may be altered in NHL, but the suite of targets and the effect of individual MEF2B mutations on the activity of these proteins remains to be elucidated ( Figure 3 ). 10

gr3

Figure 3 Patterns of mutations observed in newly identified NHL-related genes. Shown are all somatic mutations in the COSMIC database 53 detected in lymphoid cancers from all published studies (as of April 2013). EZH2 was the first lymphoma-related gene identified by NGS. In lymphomas, mutations in this gene are almost entirely restricted to codon 641, which encodes a tyrosine residue (Y641). This mutation was later demonstrated to enhance the activity of the enzyme in cooperation with wild-type EZH2.54 and 55 Both CREBBP and MLL2 demonstrate a more diffuse pattern including many small indels resulting in frameshifts. Few recurrent variants are observed in these genes with those appearing in CREBBP almost exclusively restricted to the histone lysine acetyltransferase (KAT11) domain.

Mutations in certain histone modifiers extend well beyond the GCB lymphomas. Using WGS and RNA-seq, MLL2 was found to be a novel mutation target that is mutated in up to 89% of FLs and is mutated in up to 33% of with mutations equally distributed between ABC and GCB DLBCL, making it the most commonly mutated gene in each of these cancers. 47 Consistent with tumor-suppressor function, the mutations affecting MLL2 were almost exclusively truncating or frameshift mutations ( Figure 3 ). Separate groups have since confirmed this observation by targeted sequencing of the MLL2 locus in large cohorts. 27 MLL2 encodes a histone methyltransferase responsible for methylating H3K4. Since its report, mutations in other MLL-family genes have been observed in DLBCL (namely, MLL, MLL3, and MLL5) and other histone modifiers such as HDAC7, a histone deacetylase and KDM2B, a H3K36-specific methyltransferase. Based on the relative scarcity of mutations in these published in subsequent studies, these appear to be less common targets of mutation compared to MLL2. Although the role of these mutations on epigenetic regulation of gene expression has not been definitively determined, these observations suggest the importance epigenetic alterations in the pathogenesis of NHL (reviewed in detail elsewhere 48 ). Importantly, recurrent mutations in genes involved in histone modification point to an economy for tumor cells, whereby mutational events in select genes can result in massive changes in the expression of genes by altering their epigenetic regulation.

Notch Signaling

Mutations that deregulate Notch signaling were first identified as drivers of T-cell acute lymphoblastic leukemia (ALL) 49 but NGS profiling has provided evidence that Notch signaling can be activated by similar mechanisms in many subtypes of lymphoma. WGS and exome sequencing of a small number of CLLs facilitated the first identification NOTCH1 as a recurrent mutation target in an NHL with an enrichment of mutations among the IGHV-unmutated (pre-germinal center) cases. 22a Similarly, RNA-seq analysis of mantle cell lymphoma provided the evidence for significant levels of NOTCH1 mutations in this disease. 11 In each of the cited studies, the mutations included truncations, frameshifts, and PEST domain mutations similar to those observed in T-ALL. Subsequently, a similar mutation pattern was observed in NOTCH2 in splenic marginal zone lymphoma by exome and WGS analysis ( Figure 1 ).50 and 52 This indicates that deregulated Notch signaling is common in these diseases but can arise by separate mechanisms. Interestingly, a survival comparison between mutations deregulating Notch activity showed a significant survival correlate in each of these diseases. In each disease, the presence of a mutation in the disease-relevant Notch gene correlated with adverse clinical outcome.11, 50, and 51 Collectively, mutations in these two genes can be considered a potential new prognostic biomarker for these NHLs and suggest that alternative therapeutic options for patients harboring these mutations should be explored.

Conclusion

In the last few years, the lymphoma research community has greatly benefited from the application of NGS to the discovery of novel targets of mutation. These include examples of certain genes mutated in the majority of patients with a particular subtype, such as MLL2 (in FL) and MYD88 (in LPL). As research progresses, some of these new players, MYD88 included, have been identified in additional NHL types. Some of the commonalities between genes mutated in multiple diseases reflect shared pathways and common cells of origin. Many diseases also harbor completely disease-restricted mutations, which may indicate the unique molecular events that result in these distinct clinicopathological entities. Overall, the exciting findings resulting from the study of lymphomas using NGS continue to emerge at a rapid pace and offer the potential for new prognostic tests and novel therapeutic strategies for these diseases.

Acknowledgments

R.D.M. is supported by a British Columbia Cancer Foundation Investigator Establishment Award. R.D.G. is supported by a Terry Fox New Frontiers in Cancer Program project award (# 019001).

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Footnotes

a Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada

b Genome Sciences Centre, British Columbia Cancer Agency. Vancouver, British Columbia, Canada

c Centre for Lymphoid Cancer, British Columbia Cancer Agency. Vancouver, British Columbia, Canada

d Department of Pathology, University of British Columbia. Vancouver, British Columbia, Canada

lowast Address correspondence to Ryan D. Morin, PhD, Simon Fraser University, 8888 University Dr, SSB 7157, Burnaby, BC Canada V5A 1S6

Conflicts of interest: none.