Impact of Genetics on Mature Lymphoid Leukemias and Lymphomas

Abstract

Recurrent genetic aberrations have long been recognized in mature lymphoid leukemias and lymphomas. As conventional karyotypic and molecular cloning techniques evolved in the 1970s and 1980s, multiple cytogenetic aberrations were identified in lymphomas, often balanced translocations that juxtaposed oncogenes to the immunoglobulin (IG) or T-cell receptor (TR) loci, leading to dysregulation. However, genetic characterization and classification of lymphoma by conventional cytogenetic methods is limited by the infrequent occurrence of recurrent karyotypic abnormalities in many lymphoma subtypes and by the frequent difficulty in growing clinical lymphoma specimens in culture to obtain informative karyotypes. As higher-resolution genomic techniques developed, such as array comparative genomic hybridization and fluorescence in situ hybridization, many recurrent copy number changes were identified in lymphomas, and copy number assessment of interphase cells became part of routine clinical practice for a subset of diseases. Platforms to globally examine mRNA expression led to major insights into the biology of several lymphomas, although these techniques have not gained widespread application in routine clinical settings. With the advent of next-generation sequencing (NGS) techniques in the early 2000s, numerous insights into the genetic landscape of lymphomas were obtained. In contrast to the myeloid malignancies, most common lymphomas exhibit an at least somewhat mutationally complex genome, with few single driver mutations in the majority of patients. However, many recurrently mutated pathways have been identified across lymphoma subtypes, informing targeted therapeutic approaches that are beginning to make meaningful changes in the treatment of lymphoma. In addition to the ability to identify possible therapeutic targets, NGS techniques are highly amenable to the tracking of residual lymphoma following therapy, because of the presence of unique genetic “fingerprints” in lymphoma cells due to V(D)-J recombination at the antigen receptor loci. This review will provide an overview of the impact of novel genetic technologies on lymphoma classification, biology, and therapy.

OVERVIEW OF GENETIC EVENTS IN SELECTED LYMPHOMAS

Lymphoid neoplasms are clonal proliferations of B cells, T cells, or natural killer (NK) cells that may manifest clinically as lymphadenopathy, extranodal tumors, leukemia, or any combination of the three. B-cell lymphomas comprise more than 30 World Health Organization (WHO)-defined entities and account for the majority of all lymphomas (Swerdlow et al. 2017). Different types of B-cell lymphoma are thought to originate from different stages in normal B-cell development (Fig. 1). Certain genetic events are shared in different lymphomas with similar cells of origin (e.g., mutations of chromatin modifiers in germinal center–derived B-cell lymphomas, B-cell receptor [BCR] pathway activation in postgerminal center B-cell lymphomas). T-cell lymphomas are much rarer than B-cell lymphomas, but they are frequently aggressive. Like B-cell lymphomas, some T-cell lymphomas exhibit phenotypic characteristics of physiologic T-cell subsets (Fig. 2). A comprehensive account of lymphoma genetics is outside of the scope of this review; this section will give an overview of genetic events in selected B-cell and T-cell lymphoma entities.

Figure 1.

Simplified schematic of B-cell development and the lymphomas thought to originate from specific B-cell developmental stages. Representative frequent and/or clinically relevant genetic alterations are also noted. (MCL) Mantle cell lymphoma, (BL) Burkitt lymphoma, (HGBL) high-grade B-cell lymphoma, (DLBCL) diffuse large B-cell lymphoma, (GCB) germinal center B-cell, (ABC) activated B-cell, (FL) follicular lymphoma, (CLL/SLL) chronic lymphocytic leukemia/small lymphocytic lymphoma, (HCL) hairy cell leukemia, (HCL-v) hairy cell leukemia-variant, (SMZL) splenic marginal zone lymphoma, (LPL) lymphoplasmacytic lymphoma.

Figure 2.

Simplified schematic of CD4⁺ T-cell subsets and selected corresponding lymphoma subtypes. Because of T-cell plasticity, the association of some of these entities with the subset is somewhat tenuous. Representative frequent and/or clinically relevant genetic alterations are also noted. (ALCL) Anaplastic large cell lymphoma, (MF/SS) mycosis fungoides/Sézary syndrome, (ATLL) adult T-cell leukemia/lymphoma, (AITL) angioimmunoblastic T-cell lymphoma, (T-PLL) T-cell prolymphocytic leukemia, (Th1) T helper type 1, (Th2) T helper type 2, (Treg) regulatory T cell, (Tfh) follicular helper T cell, (Tcm) central memory T cell.

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma

Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is the most common lymphoid leukemia. CLL/SLL is diagnosed based on characteristic morphologic, clinical, and immunophenotypic characteristics (Rawstron et al. 2018), but it has become clear that the underlying genetics of CLL/SLL are prognostically and therapeutically critical. CLL/SLL may be subdivided into two primary genetic subgroups that each constitute about one-half of cases: those cases that exhibit an unmutated immunoglobulin heavy chain (IGH), and those that harbor a somatic hypermutation of IGH (Damle et al. 1999; Hamblin et al. 1999). These differences reflect whether the founding neoplastic lymphocyte progressed through a germinal center reaction, eliciting somatic hypermutation through the effect of activation-induced cytidine deaminase (AICDA), or whether the tumor arose either from an antigen-naive lymphocyte or one that encountered antigen through a germinal center–independent mechanism. This genomic distinction has major clinical implications, as patients with unmutated CLL/SLL have worse outcomes than do those with mutated CLL/SLL (Byrd et al. 2006).

CLL/SLL commonly exhibits recurrent structural chromosomal alterations that are amenable to clinical detection by fluorescence in situ hybridization (FISH) and microarray platforms (Döhner et al. 2000). Deletions of 13q are the most common structural alteration in CLL/SLL, occurring in ∼55% of patients. 13q deletions encompass MIR15A and MIR16-1, microRNAs that are important regulators of the antiapoptotic proteins BCL2 and MCL1 (Calin et al. 2008). When present in isolation, patients with 13q deletions have a relatively favorable prognosis. Trisomy 12 is relatively common, although its identification does not lead to specific clinical intervention. It is often associated with atypical phenotypic and/or morphologic characteristics, and so its identification may be diagnostically reassuring in a somewhat unusual case. Deletions of 11q23 (targeting ATM and surrounding genes) and 17p (targeting TP53) are associated with adverse prognosis in CLL/SLL. TP53 loss is especially adverse, and its identification has historically been used to direct younger patients to transplant preparation and older patients to agents such as alemtuzumab, as 17p-deleted CLL/SLL patients exhibited especially poor responses to conventional chemoimmunotherapeutic regimens (Gribben 2010; Brown 2018). A new generation of targeted agents such as inhibitors of Bruton tyrosine kinase (BTK), phosphoinositide 3-kinase (PI3K)-δ, and B-cell leukemia/lymphoma 2 (BCL2) have clinical efficacy against this aggressive subset.

Next-generation sequencing (NGS) has allowed identification of numerous recurrently mutated genes in CLL/SLL; although similar to many other lymphomas, there is a long tail of mutated genes, and no single gene is mutated in more than one-half of cases (Puente et al. 2011; Wang et al. 2011; Quesada et al. 2012; Landau et al. 2015). Among the more commonly mutated genes are SF3B1 (in ∼20%), ATM (frequently associated with 11q deletion), TP53 (frequently associated with 17p deletion), and NOTCH1 (associated with unmutated IGHV). Identification of TP53 mutations is critical, as they impart a poor prognosis, as does 17p loss, and ∼30%–40% of patients with TP53 mutation do not have 17p losses that are identified by routine FISH testing (Malcikova et al. 2018).

All patients with CLL/SLL should be screened for mutations in TP53 given its prognostic and therapeutic significance, and FISH studies to identify recurrent copy number changes (such as 17p deletion) are necessary at diagnosis and progression. The mutational status of the CLL should be tested when a patient requires treatment, as fit patients with mutated CLL and without 17p del/TP53 mutation may exhibit excellent responses to FCR chemotherapy, although alternate approaches are needed for other patients (Eichhorst et al. 2016; Fischer et al. 2016). Ibrutinib has recently been approved as frontline therapy for CLL/SLL in all genetic types; however, ibrutinib resistance can arise in a subset of patients. Some relapses following ibrutinib therapy are mediated by acquired mutations in BTK, the target of ibrutinib, or PLCG2, which allows for BTK-independent BCR activation (Woyach et al. 2014). Screening for these mutations may be considered for patients with CLL who lose response to ibrutinib.

Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is a relatively uncommon B-cell lymphoma that, similar to CLL/SLL, typically exhibits expression of the T-cell-associated antigen CD5. Although most often a morphologically indolent-appearing lymphoma, MCL generally exhibits a progressive disease course and is incurable with conventional chemoimmunotherapeutic approaches. MCL is characterized by translocations between chromosomes 11 and 14, causing deregulation of the cyclin D1 (CCND1) gene by the IGH enhancer region. The identification of this rearrangement in most cases of MCL contributed to its recognition and acceptance as a distinct lymphoma entity (Banks et al. 1992).

Copy number changes of 13q14, 11q23, and 17p are relatively frequent events in mantle cell lymphoma, targeting regions similar to those lost in CLL/SLL (Beà et al. 1999; Bentz et al. 2000). However, mantle cell lymphomas more frequently manifest more complex karyotypes than are seen in CLL/SLL, although there is no clear clinical indication to routinely test for these copy number changes in MCL. NGS studies have identified recurrent mutations in CCND1, ATM, KMT2D (MLL2), and TP53 (Beà et al. 2013; Zhang et al. 2014). TP53 mutations, in particular, have been associated with poorer outcomes in MCL (Eskelund et al. 2017).

Although CCND1 translocations are the genetic hallmark of MCL, lymphomas with morphologic and immunophenotypic characteristics of MCL but without detectable CCND1 translocation or protein expression have been recognized (Yatabe et al. 2000). These so-called CCND1⁻ MCL have a gene expression profile similar to that seen in conventional MCL and consistently overexpress SOX11 (Fu et al. 2005; Mozos et al. 2009). SOX11 is a member of the SOX (sex determining region Y-related HMG-box) proteins and is expressed in ∼90% of MCLs, including in early “in situ” disease and in cyclin D1-negative cases (Dictor et al. 2009; Carvajal-Cuenca et al. 2012). It is now recognized that 50% of the cyclin D1-negative cases harbor CCND2 translocations resulting in juxtaposition with the immunoglobulin genes (Salaverria et al. 2013; Kurita et al. 2016; Martín-Garcia et al. 2018). Additionally, CCND3 translocations to the immunoglobulin light chain enhancers have also recently been recognized in cyclin D1-negative mantle cell lymphomas (Martín-Garcia et al. 2018).

Although most cases of MCL have a progressive disease course, some cases with a predominantly extranodal disease distribution have been recognized with more indolent clinical behavior. These cases have CCND1 translocations, tend not to express SOX11, and commonly have hypermutated IGH (as opposed to most MCL with unmutated IGH) (Fernàndez et al. 2010). This disease has been codified in the WHO classification as “leukemic non-nodal MCL.” Although generally indolent, some patients with leukemic non-nodal MCL exhibit disease progression, often associated with acquisition of 17p/TP53 alterations (Carvajal-Cuenca et al. 2012).

Hairy Cell Leukemia/Splenic Lymphomas

Hairy cell leukemia (HCL) is a rare and clinically indolent mature B-cell leukemia/lymphoma that preferentially involves the spleen, blood, and bone marrow. As opposed to most mature B-cell leukemias that typically present with lymphocytosis, HCL typically manifests with cytopenias, with relatively few circulating neoplastic lymphocytes. HCL cells have characteristic morphologic features and immunophenotypic characteristics (Shao et al. 2013). Until the development of NGS techniques, the genetic basis of HCL was unclear, as few recurrent structural alterations were present in the HCL genome (Forconi et al. 2008). However, in 2011, activating BRAF Val600Glu mutations were identified in nearly all cases of HCL (Tiacci et al. 2011). BRAF mutations are exceptionally uncommon in other B-cell lymphomas, suggesting this is a relatively specific diagnostic marker for HCL (Tiacci et al. 2011; Arcaini et al. 2012). Rare BRAF-negative HCL cases often exhibit preferential usage of IGHV4-34 and frequently have downstream mutations in MAP2K1 (Waterfall et al. 2014). BRAF-mutated HCL may also exhibit loss of wild-type BRAF through deletions, along with infrequent mutations of KMT2C and CDKN1B (Durham et al. 2017).

MAP2K1 mutations are also present in ∼30% of HCL variant (HCL-v) cases (Waterfall et al. 2014), a neoplasm with morphologic and immunophenotypic features similar to but distinct from classic HCL, and with poorer responses to single-agent purine analogs. HCL-v cases additionally exhibit recurrent mutations in CCND3, U2AF1, ARID1A, and TP53 (Waterfall et al. 2014; Durham et al. 2017). The finding of activated MAPK pathways in both HCL and HCL-v unify the pathobiology of these lymphoid leukemias to some degree and point to therapeutic targets in these diseases. Although HCL is relatively well managed by purine analogs, this is not the case for HCL-v. BRAF inhibition has been used successfully in HCL patients in the refractory/relapsed setting (Tiacci et al. 2015), and MEK inhibition has been anecdotally successful in HCL-v (Andritsos et al. 2018).

Splenic Marginal Zone Lymphoma

Splenic marginal zone lymphoma (SMZL) is a low-grade B-cell lymphoma that, like HCL and HCL-v, preferentially involves the blood, bone marrow, and spleen. SMZLs exhibit common karyotypic alterations, with frequent gains of 3 and 3q, gains of 12q, and deletions of 7q and 6q (Salido et al. 2010). NOTCH2 and KLF2 are the most frequently mutated genes in SMZL (Kiel et al. 2012; Rossi et al. 2012; Clipson et al. 2015). Both proteins are important regulators of normal marginal zone formation, and they are preferentially mutated in SMZL versus other low-grade B-cell lymphomas. NOTCH2 and TP53 mutations are associated with decreased treatment-free survival and overall survival, respectively, in SMZL (Parry et al. 2015; Campos-Martín et al. 2017).

Lymphoplasmacytic Lymphoma

Lymphoplasmacytic lymphoma (LPL) is an indolent B-cell lymphoma typically associated with marrow involvement, some degree of plasmacytic differentiation, and IgM monoclonal gammopathy. Waldenström macroglobulinemia (WM) is defined by the presence of an IgM paraprotein, with >10% marrow involvement by LPL. WM/LPL tumors frequently exhibit loss of 6q, deleting TNFAIP3 (Braggio et al. 2009). NGS studies identified MYD88 Leu265Pro mutations in ∼90% of patients with LPL (Treon et al. 2012). MYD88 Leu265Pro mutations are relatively uncommon in other low-grade B-cell lymphomas, although they do rarely occur in SMZL and CLL. Both activating mutations in MYD88 and deletions of TNFAIP3 lead to activation of the NF-κΒ signaling pathway. Patients with MYD88-mutated LPL appear very sensitive to BTK inhibition with ibrutinib, but efficacy is diminished in patients with concurrent mutations of CXCR4 and in the rare MYD88-wild type cases (Treon et al. 2015; Dimopoulos et al. 2017).

Follicular Lymphoma

Follicular lymphoma (FL) is the most common indolent B-cell lymphoma in western countries, accounting for ∼20% of all lymphomas worldwide (The Non-Hodgkin's Lymphoma Classification Project 1997). It is composed of germinal center–derived B cells and frequently exhibits a follicular growth pattern. Its genetic hallmark is the translocation of chromosomes 14 and 18, causing the antiapoptotic gene BCL2 to be up-regulated by the IGH enhancer, similar to the CCND1 rearrangement seen in MCL (Fukuhara et al. 1979; Tsujimoto et al. 1984). IGH/BCL2 is by itself insufficient to cause lymphoma (Roulland et al. 2006) and requires the presence of additional genetic aberrations. In addition to the t(14;18), FL harbors frequent losses of 1p, 6q, 10q, and 17p, with gains of 1q, 6q, 7, 8q, 12q, 17q, and 18 (Cheung et al. 2009). FL contains several highly recurrent mutations in addition to IGH/BCL2 fusion. Mutations in chromatin modifiers are particularly frequent, with KMT2D, CREBBP, EZH2, and EP300 being common mutational targets (Morin et al. 2010, 2011; Pasqualucci et al. 2011). Mutations of chromatin modifiers are present in >90% of FLs, and >70% have mutations in two or more chromatin-modifying genes (Green et al. 2015). In addition to its frequent deletion at chromosome 1p in FL, TNFRSF14 is commonly mutationally inactivated, and TNFRSF14 alterations are present in more than one-half of FL cases (Cheung et al. 2010; Launay et al. 2012). Loss of TNFRSF14 leads to B-cell proliferation through increased BCR signaling and leads to changes in the FL microenvironment that facilitate tumor growth (Boice et al. 2016).

Although FL is an indolent lymphoma, it is generally incurable with conventional immunochemotherapeutic approaches, and transformation to an aggressive B-cell lymphoma is a frequent and clinically devastating event. Several recent studies have explored the underlying genetic basis of transformation in follicular lymphoma. Transformed FL (t-FL) appears to arise from an ancestral clone shared with the dominant FL, rather than as a direct progression of the dominant FL clone. t-FL is enriched for alterations of MYC, CDKN2A/B, TP53, PIM1, EBF1, and B2M (Elenitoba-Johnson et al. 1998; Pasqualucci et al. 2014; Okosun et al. 2014).

A modification of the standard FL international prognostic index (FLIPI) has been proposed that incorporates the presence of mutations in seven commonly mutated genes (m7-FLIPI) (Pastore et al. 2015). In this prognostic model, EZH2, ARID1A, and MEF2B mutations were favorable (particularly EZH2), whereas mutations in EP300, FOXO1, CREBBP, and CARD11 were unfavorable (particularly EP300 and FOXO1). The m7-FLIPI model improved on standard FLIPI risk stratification in FL patients by identifying patients with high-risk FLIPI scores but with favorable outcomes, largely associated with EZH2 mutation.

Pediatric-type FL (PTFL) is an uncommon and highly indolent lymphoma of germinal center B cells that typically occurs in adolescent males. In contrast to conventional FL, PTFL is less genomically complex, with significantly fewer aberrations and without translocations of BCL2 or BCL6. Like FL, PTFL exhibits frequent losses of 1p/TNFRSF14 mutations, but mutations of chromatin modifiers are virtually absent, whereas MAP2K1 mutations are relatively frequent (Louissaint et al. 2016; Schmidt et al. 2016).

Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma worldwide (The Non-Hodgkin's Lymphoma Classification Project 1997). The term DLBCL encompasses a genetically diverse group of neoplasms with the common feature of a diffuse proliferation of large, transformed B cells. The heterogeneity of DLBCL was recognized in lymphoma classification systems for decades, but subclassification based on morphologic features is not reproducible among experts and does not clearly associate with outcomes (Harris et al. 1994). In 2000, a landmark study demonstrated that DLBCL could be subdivided into clinically relevant categories based on gene expression as assessed by cDNA microarray analysis (Alizadeh et al. 2000). One large group of DLBCLs exhibited a gene expression profile resembling that of germinal center B cells (GCBs), whereas another exhibited a profile similar to that of activated peripheral blood B cells (ABCs). Some cases remained unclassifiable by this approach. Patients with GCB-DLBCL had better outcomes than patients with ABC-DLBCL when treated with CHOP (Rosenwald et al. 2002), and the survival differences persisted with the addition of rituximab to the CHOP regimen (Lenz et al. 2008). Assessment of gene expression signatures has not historically been clinically feasible, and so surrogate immunohistochemical algorithms were developed and implemented clinically (Hans et al. 2004). Newer gene expression technologies, however, hold the promise of robust distinction between ABC- and GCB-DLBCL in routinely processed formalin-fixed, paraffin-embedded material (Scott et al. 2014).

The GCB/ABC paradigm has informed subsequent genetic exploration of DLBCL. Both ABC and GCB subsets exhibit relatively frequent translocations of BCL6 and mutations of KMT2D, CREBBP, EP300, B2M, TP53, and MEF2B; GCB DLBCL exhibit frequent IGH/BCL2 translocation, mutations of EZH2, and MYC translocations; and ABC DLBCL often have alterations of TNFAIP3, CDKN2A/B, MYD88, PRDM1, CD79A/B, CARD11, and frequently exhibit amplification (rather than translocation) of BCL2 (for review, see Pasqualucci and Dalla-Favera 2015). Many of the mutations identified in ABC-DLBCL lead to chronic active BCR signaling and NF-κB activation, pointing to targetable pathways in this chemoresistant disease (Davis et al. 2010; Ngo et al. 2011).

Recent studies have proposed classifications of DLBCL based on their mutational signatures (Chapuy et al. 2018; Schmitz et al. 2018). Although the studies exhibited some differences in their findings, several common themes emerge, emphasizing that the biologic and clinical heterogeneity of DLBCL cannot be captured with a trichotomous ABC/GCB/other gene expression classification. In both studies, two subgroups were recognized in ABC-DLBCL: One had translocations of BCL6 and mutations of genes in the NOTCH2 pathway, suggesting a possible shared cell of origin with MZL, as splenic MZLs frequently harbor NOTCH2 pathway mutations (Kiel et al. 2012; Rossi et al. 2012) and transformed gastric extranodal MZLs frequently have rearrangements of BCL6 (Chen et al. 2006; Flossbach et al. 2011).This group was associated with relatively favorable survival in both studies and was also enriched in cases with an unclassifiable gene expression profile (neither clearly ABC or GCB). The other ABC cohort consisted of lymphomas with MYD88 Leu265Pro mutations and mutations of CD79B. This group had an unfavorable prognosis and was enriched for lymphomas with extranodal involvement, concordant with results of previous studies that have identified frequent mutations of MYD88 in primary extranodal lymphomas such as primary CNS lymphoma, testicular DLBCL, and primary cutaneous DLBCL, leg-type (Pham-Ledard et al. 2014; Chapuy et al. 2016). In both studies, a group of GCB-DLBCL characterized by BCL2 mutation/rearrangement and mutation of EZH2 was identified with worse survival than other GCB-DLBCLs. The remaining GCB cases were further characterized by Chapuy and colleagues as commonly having mutations in four linker and four core histone genes, along with CD83, CD58, CD70, RHOA, GNA13, SGK1, CARD11, NFKBIE, NFKBIA, BRAF, and STAT3, without mutations in chromatin modifiers. Chapuy and colleagues also recognized a TP53-mutated cohort with multiple structural chromosomal abnormalities, and Schmitz et al. recognized a small cohort of NOTCH1-mutated ABC DBLCL with adverse outcomes.

These efforts point to the biologic complexity of a category that is still classified primarily by its morphologic appearance and that is still treated homogeneously at diagnosis. Although frontline immunochemotherapy for DLBCL has not changed significantly since the introduction of rituximab in the early 2000s, novel agents are now routinely used in the in the relapsed/refractory setting. Particularly favorable responses have been seen in relapsed/refractory ABC DLBCL to agents such as ibrutinib and lenalidomide (Wilson et al. 2015; Czuczman et al. 2017). The specific mutations present in an individual case of DLBCL may better predict response to targeted therapies than the overall gene expression profile, as responses to ibrutinib in ABC DLBCL appear enriched in MYD88/CD79B comutated DLBCL cases (Wilson et al. 2015). The genetic heterogeneity that has been uncovered in DLBCL should inform clinical trials going forward so that possible responses in particular genomic subgroups are not lost.

Burkitt Lymphoma

Burkitt lymphoma (BL) is a highly aggressive mature B-cell neoplasm that arises in different epidemiologic scenarios. Endemic BL is highly associated with EBV infection and occurs in areas with endemic malaria; sporadic BL occurs worldwide and is less frequently associated with EBV; and immunodeficiency-associated BL occurs in patients with immunodeficiency, most commonly HIV infection (Swerdlow et al. 2017). BL is highly aggressive, but curable with intensive chemoimmunotherapy, and its distinction from DLBCL is paramount in adults.

BL is the prototypical example of a lymphoma with a recognized genetic abnormality: extra material on chromosome 14 was first recognized in BL in 1972 (Manolov and Manolova 1972). This material was soon characterized as originating from chromosome 8 (Zech et al. 1976), and the IGH/MYC rearrangement was described soon after (Dalla-Favera et al. 1982; Taub et al. 1982). All epidemiologic types of BL are highly associated with translocations of MYC to immunoglobulin loci, with only rare MYC-fusion-negative cases that may have MYC deregulation by alternate mechanisms (Hummel et al. 2006; Leucci et al. 2008). MYC translocations are not specific to BL, however, as they are present in a minor subset of DBLCL and in many non-BL high-grade B-cell lymphomas (discussed below). Therefore, accurate diagnosis requires integration of genetic, morphologic, and phenotypic findings. Molecular studies may help distinguish between BL and DLBCL, as aside from MYC translocations, BL exhibits few structural alterations, in contrast to DLBCL (Hummel et al. 2006). Sporadic cases of BL exhibit high-frequency mutations of TCF3 or its negative regulator ID3, and oncogenic mutations of CCND3 are common (Schmitz et al. 2012). EBV-associated endemic BL, in contrast, exhibits less frequent mutations of TCF3, ID3, and CCND3, with more frequent mutations of ARID1A, RHOA, and CCNF (Schmitz et al. 2012; Abate et al. 2015). Many genes commonly mutated in DLBCL are only rarely mutated in BL, such as EZH2, SGK1, BCL2, CD79B, and MYD88.

High-Grade B-Cell Lymphomas with MYC and BCL2 and/or BCL6 Rearrangements

High-grade B-cell lymphoma (HGBL) with MYC and BCL2 and/or BCL6 rearrangements is a category introduced in the 2016 revision of the WHO Classification that encompasses cases that previously would have been classified as “B-cell lymphoma, unclassifiable, with features intermediate between DLBCL and BL.” As the name suggests, this category is intended to encompass all mature, aggressive-appearing lymphomas that harbor translocations of MYC, along with a translocation of one or both of BCL2 and BCL6. These lymphomas are commonly referred to as “double-hit” lymphomas (DHLs) and have been associated with very poor outcomes when treated with conventional DLBCL therapy (Johnson et al. 2009; Li et al. 2012). They may have Burkitt-like, blastoid, or DLBCL-like morphology. The introduction of this category and its clinical implications has mandated FISH testing for these fusions in most, if not all, cases of large B-cell lymphoma, as strategies that FISH only cases with a GCB phenotype and MYC/BCL2 protein expression miss a large percentage of rearranged cases (Scott et al. 2018). Selection of FISH probes impacts sensitivity of MYC rearrangement detection as breaks may occur over several megabases. The most comprehensive methods would rely on application of both break-apart and dual-fusion FISH probes (May et al. 2010; Muñoz-Mármol et al. 2013), although this approach is resource-intensive. Several groups have reported that the MYC translocation partner may be prognostically significant, and that lymphomas with nonimmunoglobulin (non-IG) MYC translocation partners have better outcomes than those with IG/MYC fusions (Johnson et al. 2009; Pedersen et al. 2014; Copie-Bergman et al. 2015; Chong et al. 2018), further complicating possible FISH analysis. The most appropriate regimen for DHL is not clear, but patients benefit from regimens more intensive than standard R-CHOP therapy (Friedberg 2017).

Primary Mediastinal (Thymic) Large B-Cell Lymphoma and Classic Hodgkin Lymphoma

Primary mediastinal (thymic) large B-cell lymphoma (PMBL) is an uncommon genetically distinct subtype of large B-cell lymphoma that occurs in the mediastinum, often in young females. Although it exhibits more robust B-cell antigen expression, PMBL exhibits some morphologic, immunophenotypic, and genetic similarity to classic Hodgkin lymphoma (CHL), which also commonly occurs in the mediastinum in younger patients. Early gene expression profiling studies identified PMBL as distinct from DLBCL more generally, and with shared gene expression features with CHL (Rosenwald et al. 2003; Savage et al. 2003). Later mutational studies have confirmed additional shared features with CHL, including 9p24.1 amplifications involving JAK2, CD274 (PDL1) and PDCD1LG2 (PDL2) (Green et al. 2010); CIITA fusions (Steidl et al. 2011); SOCS1 inactivation (Melzner et al. 2005; Weniger et al. 2006); and STAT6 mutations (Ritz et al. 2009; Tiacci et al. 2018). Recent studies have identified IL4R mutations as another activator of JAK-STAT signaling in PMBL (Viganò et al. 2018a), and these mutations are also present in some cases of DLBCL (Viganò et al. 2018b). Nonmediastinal PMBL have been described, yet they are difficult to confidently diagnose (Yuan et al. 2015). As PMBL is often treated more aggressively than most DLBCLs (Dunleavy et al. 2013), recognition of nonmediastinal PMBL could have clinical implications. Mutational analysis or newer gene expression techniques may assist with this rare diagnosis and could be helpful to support the impression of PMBL when diagnosing a large B-cell lymphoma in the mediastinum (Chong et al. 2018).

Genetic studies of CHL have been historically limited by the relative paucity of the malignant Hodgkin Reed–Sternberg (HRS) cells in most biopsy specimens. However, novel techniques have allowed single-cell interrogation of HRS cells, revealing a mutational profile with significant overlap with PMBL, as discussed above. Copy number alterations of the PDL1/PDL2 locus are particularly common in CHL, occurring in nearly every lymphoma in one series (Roemer et al. 2016). Amplifications (as opposed to polysomy or low-level copy gain) of PDL1/PDL2 were associated with decreased progression-free survival and advanced-stage disease in this study and are a likely mechanism for the encouraging clinical responses to immune checkpoint blockade seen in CHL (Ansell et al. 2015). Although relatively few cases of EBV⁺ CHL have been comprehensively genetically studied, they appear to have a lower mutational burden than EBV^– CHL and contain fewer copy number alterations (Montgomery et al. 2016; Tiacci et al. 2018).

T-Cell Lymphomas

Anaplastic Large Cell Lymphoma

Anaplastic large cell lymphoma (ALCL) is a CD30⁺ mature T-cell lymphoma that is divided into two primary groups on the basis of its genetic abnormalities: ALCL, ALK-positive (ALK⁺ ALCL) and ALCL, ALK-negative (ALK⁻ ALCL). ALK⁺ ALCL is characterized by the fusion of the tyrosine kinase ALK to other genes, most commonly NPM1 (Le Beau et al. 1989; Rimokh et al. 1989; Morris et al. 1994). This leads to aberrant expression of the ALK protein, easily identifiable by immunohistochemical staining (Pittaluga et al. 1997; Pulford et al. 1997; Falini et al. 1998). ALK expression activates multiple signaling pathways, including JAK-STAT, PI3K-AKT, mTOR, MAPK, and others (for review, see Hallberg and Palmer 2013). Small-molecule inhibitors of ALK, such as crizotinib, have demonstrated clinical efficacy in ALK⁺ ALCL (Mossé et al. 2017).

ALK⁻ ALCL, by definition, lacks ALK rearrangement or overexpression. In comparison with ALK⁺ ALCL, ALK⁻ ALCL tend to exhibit a somewhat more complex genome, with a higher frequency of chromosomal gains and losses (Boi et al. 2013). ALK⁻ ALCL exhibits a heterogeneous clinical course, and emerging evidence demonstrates that outcomes are heavily dependent on the underlying lymphoma genetics. DUSP22 rearrangements are present in ∼30% of ALK⁻ ALCLs, including cutaneous forms of ALCL (Feldman et al. 2009, 2011; Parrilla Castellar et al. 2014). More rarely, TP63 rearrangements occur in ALK⁻ ALCL (Vasmatzis et al. 2012; Parrilla Castellar et al. 2014), and together these translocations identify clinically relevant ALK⁻ ALCL subgroups. DUSP22-rearranged systemic ALK⁻ ALCL have a favorable prognosis similar to that seen in ALK⁺ ALCL, whereas TP63-rearranged cases do poorly with conventional chemotherapy (Parrilla Castellar et al. 2014; Pedersen et al. 2017). Other recurrent kinase fusions, such as those involving TYK2, occur with less frequency in ALK⁻ ALCL (Velusamy et al. 2014; Crescenzo et al. 2015) but activate JAK-STAT signaling, whereas mutations of JAK1 and/or STAT3 are present in ∼20% of cases (Crescenzo et al. 2015). Although the introduction of brentuximab vedotin (an anti-CD30 antibody-drug conjugate) has led to improved therapies in ALK⁺ and ALK⁻ ALCL (Horwitz et al. 2019), the identification of mutationally activated pathways provides alternate drug targets for therapeutic exploration.

T-Cell Prolymphocytic Leukemia

T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell leukemia that typically presents with marked lymphocytosis and is associated with poor survival. T-PLL is commonly associated with inv(14) (Malcikova et al. 2018), which juxtaposes the TRA/D locus with the oncogenes TCL1A and TCL1B (Brito-Babapulle et al. 1987; Russo et al. 1989; Pekarsky et al. 1999). This translocation is analogous to the IG translocations commonly seen in B-cell lymphomas, causing the aberrant expression of TCL1A/B owing to its localization near the TRA/D enhancer, leading to increased AKT1 signaling (Pekarsky et al. 2000). Additional common structural events in T-PLL include loss of the ATM gene at 11q23 and trisomy 8. In addition to these large-scale genomic events, point mutations in the JAK-STAT pathway are common (Kiel et al. 2014). Recurrent mutations of JAK1, JAK3, and STAT5B are present in ∼8%, ∼30%, and ∼36% of T-PLL cases, respectively. Inactivating mutations and deletions of EZH2 are additionally present in about one-half of cases (Kiel et al. 2014). Alemtuzumab, an anti-CD52 antibody, has been the mainstay of therapy for T-PLL, but this regimen is not curative and can be associated with significant complications (Dearden 2012). Alternate therapeutic approaches are therefore needed. Although JAK-STAT kinase inhibitors are clinically available, it does not appear that response to JAK-STAT inhibition is readily predictable by mutational status in ex vivo studies (Andersson et al. 2018).

Mycosis Fungoides/Sézary Syndrome

Mycosis fungoides (MF) is the most common cutaneous T-cell lymphoma (CTCL), and it typically exhibits a fairly indolent disease course. However, MF may progress to involve the blood, exhibiting clinical and pathologic overlap with Sézary syndrome (SS), which strictly defined is a de novo aggressive and rare process characterized by erythroderma, lymphadenopathy, and circulating lymphoma cells. MF harbors recurrent mutations of PLCG1 (Kiel et al. 2015), leading to activation of pathways downstream of the T-cell receptor (Vaqué et al. 2014). TP53 mutations occur in MF, more commonly in tumor-stage patients than at earlier stages (McGregor et al. 1999). MF exhibits a relatively simple genome (da Silva Almeida et al. 2015), whereas SS exhibits a highly complex genome, typically with numerous copy number changes and structural rearrangements (Choi et al. 2015; da Silva Almeida et al. 2015; Kiel et al. 2015; Wang et al. 2015b). Common mutational/deletional targets in SS include numerous genes involved in epigenetic regulation (ARID1A, ZEB1, DMNT3A, TET2, CREBBP, and KMT2D), components of several signaling pathways (CARD11, CCR4, PLCG1, BRAF, MAKP1, JAK1, JAK3, STAT3, and STAT5B) and tumor suppressors such as CDKN2A and TP53. CCR4 is a therapeutic target in CTCL, as mogamulizumab, an anti-CCR4 antibody, has recently been Food and Drug Administration (FDA)-approved in the relapsed/refractory setting (Kim et al. 2018). It is not clear that CCR4 mutational status impacts mogamulizumab response in CTCL, although CCR4 mutations sensitize to mogamulizumab therapy in the HTLV1-associated adult T-cell leukemia/lymphoma (Sakamoto et al. 2018).

Nodal T Follicular Helper T-Cell Lymphomas

A large group of nodal peripheral T-cell lymphomas exhibit phenotypic characteristics of normal follicular helper T-cells (TFHs). Angioimmunoblastic T-cell lymphomas (AITLs) and follicular T-cell lymphoma (FTCL) are the two main nodal TFH lymphoma subtypes recognized by the WHO classification (Swerdlow et al. 2017). Recent NGS studies have identified recurrent mutations in epigenetic regulators in both AITL and FTCL, contributing to the recognition that these lymphomas exhibit overlapping biologic features, in addition to a largely shared immunophenotype. Both AITL and FTCL exhibit common mutations of TET2, DNMT3A, and RHOA (Lemonnier et al. 2012; Odejide et al. 2014; Palomero et al. 2014; Sakata-Yanagimoto et al. 2014). IDH2 mutations, in contrast, seem to be restricted to AITL, present in ∼20%–30% of cases (Cairns et al. 2012; Odejide et al. 2014; Wang et al. 2015a). The IDH2 mutations in AITL seem to always target the arginine residue at codon 172, rather than the Arg140 mutations that predominate in acute myeloid leukemia (Marcucci et al. 2010), and in contrast with acute myeloid leukemia, IDH2 and TET2 mutations are not mutually exclusive in AITL (Lemonnier et al. 2012). Arg172 mutations lead to greater 2-HG accumulation than do Arg140 mutations (Ward et al. 2013), and IDH mutations identify a subset of AITL patients with increased H3K27me3 and hypermethylation of gene promotor regions compared to IDH-wild type cases (Wang et al. 2015a). Demethylating agents have demonstrated some clinical efficacy against the lymphoma in AITL patients with concurrent myeloid neoplasms, suggesting that epigenetic modification may be a promising therapeutic approach in AITL (Lemonnier et al. 2018).

Impact of Molecular and Genomic Techniques on Lymphoma Diagnosis and Monitoring

Immunoglobulin/T-Cell Receptor Sequencing

During normal development, lymphocytes undergo somatic V(D)-J recombination of their immunoglobulin (IG) or T-cell receptor (TR) genes. This process generates a genetic “fingerprint” for each unique lymphocyte, as variable numbers of nontemplated nucleotides are inserted at the junctions of the V, D, and J genes, leading to the normal IG and TR repertoire necessary for normal antigen recognition. As lymphomas arise from a single progenitor cell, they exhibit a common, dominant V(D)-J rearrangement, as opposed to a multitude of rearrangements in reactive lymphoproliferations. This phenomenon has long been exploited in the diagnosis of lymphoma, and the detection of monoclonality through assessment of IG or TR diversity through Southern blotting or polymerase chain reaction (PCR) techniques has been a routine ancillary diagnostic technique in hematopathology for decades (van Dongen and Wolvers-Tettero 1991; van Dongen et al. 2003). Routine PCR techniques for monoclonality assessment retain advantages in turnaround time, cost, and ease of analysis. However, using typical capillary electropherograms, the amplified PCR products are separated solely on the basis of size, and each peak in the electropherogram represents the integrated signal of potentially many clones. Although this is not a problem in most cases, this phenomenon can make it difficult to always compare clones between specimens with great confidence and may lead to false-positive calls. Next-generation sequencing (NGS) techniques, on the other hand, generate the sequence itself of each individual V(D)-J read, and reads with shared sequences can be summed to give accurate quantification of each clone present in a specimen.

NGS techniques therefore allow measurement not only of whether a clone is present, but routine determination of hypermutation status and gene family usage, clinically relevant information in common diseases such as CLL/SLL (Byrd et al. 2006; Ghia et al. 2008). NGS techniques allow straightforward and definitive comparison between lymphomas in an individual patient to assess for clonal relatedness, a particularly important determination in the setting of transformation (Rossi et al. 2011). Perhaps the most powerful application of NGS for lymphoid clonality assessment is in the disease monitoring setting. Although minimal residual disease (MRD) testing can be successfully performed with conventional PCR (van der Velden et al. 2007), patient-specific primers are necessary to gain appropriate sensitivity, which is laborious and difficult to implement in most clinical laboratories. As NGS outputs the sequence of each individual read obtained in a specimen, MRD-level detection of lymphoma/leukemia-specific reads can be easily achieved with common primers as long as a sufficiently large number of reads are obtained and the sequence of the diagnostic IG or TR rearrangement is known. This technique has been used for successful disease monitoring in mature lymphomas, acute leukemias, and plasma cell myelomas (Wu et al. 2012; Kurtz et al. 2015; Roschewski et al. 2015; Scherer et al. 2016; Rossi et al. 2017; Perrot et al. 2018; Wood et al. 2018), as the only requirement is the presence of either an IG or TR rearrangement in the neoplastic cells. NGS techniques have recently been FDA-approved in the B-ALL and myeloma MRD setting, and the performance characteristics of the assays compare favorably to that of multiparameter flow cytometry in these diseases.

Clinical Genomics Testing in Lymphoma

Genetic testing in lymphoma has long been performed routinely in the clinical laboratory through comprehensive, but low-resolution, methods such as metaphase karyotyping or through targeted interrogation of specific loci such as through FISH studies. More recently, testing for individual genetic mutations has become common in some circumstances, such as testing for BRAF mutations in suspected HCL or MYD88 mutations in suspected LPL. The recent introduction of high-throughput genomic platforms into the clinical laboratory has made routine extensive genomic characterization of lymphomas feasible, and examples of clinically relevant testing are listed in Table 1.

Table 1.

Examples of genetic biomarkers and possible therapeutic implication in lymphoma entities

Disease	Genetic biomarker	Therapeutic implication
CLL	TP53 mutation/deletion	Ibrutinib therapy indication
CLL	BTK or PLCG2 mutation	Ibrutinib resistance
LPL	MYD88 mutation	Reduced ibrutinib response if MYD88 is not mutated
LPL	CXCR4 mutation	Reduced ibrutinib response if CXCR4 is mutated
HCL	BRAF mutation	Vemurafenib response
HGBL	MYC and BCL2 and/or BCL6 rearrangements	R-CHOP is suboptimal therapy
DLBCL	MYD88/CD79B comutation	Increased response to ibrutinib
FL/DLBCL	EZH2 mutation	EZH2 inhibitors in clinical trials
AITL	IDH2 mutation	IDH2 inhibitors in clinical trials
ALCL, ALK+	ALK rearrangement	May respond to crizotinib
ALCL, ALK−	DUSP22 rearrangement	May be candidate for less intensive therapy than other ALCL, ALK−

(CLL) Chronic lymphocytic leukemia, (LPL) lymphoplasmacytic lymphoma, (HCL) hairy cell leukemia, (HGBL) high-grade B-cell lymphoma, (DLBCL) diffuse large B-cell lymphoma, (FL) follicular lymphoma, (AITL) angioimmunoblastic T-cell lymphoma, (ALCL) anaplastic large cell lymphoma, (ALK⁺) ALK receptor tyrosine kinase positive, (ALK⁻) ALK receptor tyrosine kinase negative.

As discussed previously, recent studies have shown that the traditional, gene expression–based classification of DLBCL (the most common lymphoma type) into ABC and GCB subtypes is overly simplistic, with prognostically important subsets only identifiable by broad genomic profiling (Chapuy et al. 2018; Schmitz et al. 2018). Emerging data suggest that at least some of these genetic fingerprints predict for response to targeted therapies such as ibrutinib (Wilson et al. 2015), but it is likely that other factors in addition to mutational activation impact therapeutic responses, particularly when targeting pathways that are generally important in lymphoid survival (Phelan et al. 2018). As many mutations are present at relatively low frequency in most lymphoma subtypes, broad testing panels would be necessary to identify rare but potentially targetable lesions, such the infrequent but recurrent mutations of BRAF in nodal marginal zone lymphomas (Pillonel et al. 2018). Although examples of genetically directed, targeted therapy remain relatively few in lymphoma, ever-increasing numbers of novel agents are being evaluated in clinical trials, particularly in the setting of relapsed/refractory disease, and comprehensive genetic profiling of lymphomas should be considered in such settings both to identify targets and to identify the determinants of response to novel therapies.

CONCLUSION

NGS technologies have led to an exponential increase in our understanding of the pathogenesis of mature lymphoid leukemias and lymphomas. Although relatively few lymphomas had been associated with karyotypically recognizable abnormalities such as chromosomal translocations, recurrent mutations are now known in virtually every lymphoma subtype. With some exceptions, such as the very high incidence of BRAF mutations in HCL, most lymphoma types are not associated with a single highly recurrently mutated gene or recurrent translocation. Rather, lymphomas exhibit mutational complexity, with a long tail of mutations that are present in relatively few patients. However, within this genetic heterogeneity new subgroups have been recognized, possibly with divergent responses to novel therapeutic agents. Our greater understanding of mutational processes in lymphoma holds the promise of better and less toxic therapies for patients with these devastating diseases.