Ontogeny, Genetics, Molecular Biology and Classification of B and T cell Non-Hodgkin’s Lymphoma

Introduction

The advent of genome-scale profiling technologies has dramatically improved our understanding of the biological basis of mature B- and T-cell lymphomas. The purpose of this review is to place newer biological and molecular findings in the context of principles that are applicable across the spectrum of mature lymphoid malignancies.

Principles of diagnosis and classification

Lymphoid cancers are diverse in their clinical behavior and response to therapies; therefore, accurate sub-classification of lymphomas is indispensable for clinical care. Under the broadly accepted World Health Organization (WHO) classification system¹, each lymphoma subtype is defined by a unique combination of characteristic findings that may include cytological appearance, immunophenotype, microscopic growth pattern, anatomic sites of involvement as assessed by physical exam and radiological studies, genetic mutations, chromosomal rearrangements / copy number abnormalities, and other specific laboratory findings. Clinical history may also be central to the definition of certain lymphoid neoplasms, as in the case of post-transplant lymphoproliferative disorders. Evaluation of lymphoid architecture and heterogeneity are essential for lymphoma classification, and therefore an excisional lymph node biopsy should be procured whenever feasible for initial diagnosis². Accurate classification of lymphoid malignancies remains challenging, and reclassification may be anticipated in ≈20% of cases following expert hematopathology review³.

Relationship of lymphoma to normal lymphoid development

Most lymphomas recapitulate characteristic morphological, phenotypic, epigenetic, and gene expression features of specific stages in normal lymphoid cell development, although altered to varying degrees by genetic or epigenetic aberrations (Figure 1 and Table 1). For example, follicular lymphoma (FL) recapitulates the morphology and immunophenotype of B cell populations in the germinal center (GC) light zone, while marginal zone lymphomas (MZL) can show a spectrum of differentiation that resembles specific non-GC B cell and plasma cell subpopulations, and angioimmunoblastic T-cell lymphoma (AITL) recapitulates the appearance and gene expression program of T-follicular helper cells. In diffuse large B-cell lymphoma (DLBCL), major subgroups show gene expression programs characteristic of normal GC B cells (GCB-DLBCL) or in vitro activated peripheral blood B cells (ABC-DLBCL)⁴. Distinguishing these subgroups is of ongoing interest in clinical trials for targeted agents, but remains challenging in routine practice due to the imprecision of immunohistochemical classification algorithms, limited availability of more precise RNA-based classifier technologies⁵, and likelihood that additional subgroups exist^6,7.

Figure 1 –

Relationship of lymphomas to normal lymphocyte development. Stages of lymphoid development, with corresponding mature B-, T-, and NK-cell lymphoma subtypes indicated in red text. Proliferative stages of B cell development are colored violet.

HSC: hematopoietic stem cell. Lymphomas are abbreviated as indicated in Table 1.

Table 1:

Subtypes and Features of selected Non-Hodgkins Mature Lymphoid Cancers

	Lymphoma Subtype	Abbreviation	Key diagnostic features	Genomic aberrations (selected)	Suggested precursor	Clinical aggressiveness	Notes
B-cell
	Follicular lymphoma	FL	Small & large cells in follicles. CD10+, BCL6+, BCL2+(most).	IGH-BCL2, KMT2D, CREBBP, TNFRSF14, EZH2, mTOR pathway	In situ follicular neoplasia	Low (grade 1–2) to moderate (grade 3)	Low-grade FL can be partially or (rarely) entirely diffuse
	Nodal marginal zone lymphoma	NMZL	Expanded marginal zones, small/colonized follicle centers	KMT2D, NOTCH2, PTPRD, KLF2, BCR pathway	Unknown	Low to moderate	Rare and challenging to diagnose due to lack of distinctive markers
	Extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue	MALT lymphoma	Clonal small B cells and plasma cells. Lymphoepithelial lesions and follicular colonization.	BIRC3-MALT1, IGH-MALT1, BCL10-IGH, FOXP1-IGH, TNFAIP3, other BCR pathway, TP53, MYD88, NOTCH2, BCL6 R	Clonal B cells in setting of localized chronic inflammation (i.e. lymphoepithelial siladenitis)	Low	Common sites of origin include stomach, intestine, ocular adnexa, salivary glands, lung, thyroid, skin, and soft tissues
	Splenic marginal zone lymphoma	SMZL	Marrow sinusoidal infiltrate and aggregates, splenomegaly w/expanded white pulp	KLF2, NOTCH2, TRAF3, TNFAIP3, MYD88, TP53, BCR pathway	Monoclonal B-cell lymphocytosis, non-CLL-type	Low	Cells in peripheral blood may show characteristic “polar villi”
	Lymphoplasmacytic lymphoma	LPL	Plasmacytoid lymphocytes, Dutcher bodies, IgM paraprotein	MYD88 L265P (>90%), CXCR4, CD79B, KMT2D, TP53, ARID1A	IgM monoclonal gammopathy of undetermined significance	Low	Also known as Waldenstrom macroglobulinemia
	Chronic lymphocytic leukemia / small lymphocytic lymphoma	CLL / SLL	Small cells in blood with proliferation centers in nodes. CD5+, CD23+, LEF1+, FMC7-neg.	del(13q14), +12, NOTCH1, SF3B1, ATM, TP53, CHD2, BIRC3, Toll-like receptor pathway	Monoclonal B-cell lymphocytosis, CLL-type	Low (IGHV-mut) to moderate (TP53 or ATM mutated)	Early genetic events may occur in an abnormal hematopoietic stem cell clone
	Mantle cell lymphoma	MCL	Uniform small cells or blastoid. CD5+, CyclinD1+, Sox11+(most).	IGH-CCND1, UBR5, ATM, TP53, NOTCH1/2, BIRC3, TRAF2	In situ mantle cell neoplasia	Moderate, low (Sox11neg), or high (blastoid)	Rare cyclin D1-neg cases identifiable with Sox11
	Hairy cell leukemia	HCL	Cytoplasmic projections. CyclinD1+, Annexin A1+	BRAF V600E (~100%)	Abnormal hematopoietic stem cell -> clonal B cell	Moderate	Hairy cell variant has mutations in other MAP kinase genes
	Diffuse large B-cell lymphoma, germinal center B-cell subtype	GCB-DLBCL	Large cells growing in sheets. CD20+, CD10+, BCL6+, GCET1+, LMO2+.	BCL2-R, CREBBP, EZH2, KMT2D, TNFRSF14, GNA13, MEF2B, PTEN, SGK1, NFKBIA/E, REL-Amp, MYC-R	Evidence for 2 genetic subgroups, BCL2-R group may share origin with FL	High	High-grade lymphomas with concurrent MYC and BCL2 rearrangements are more aggressive & separately classified. Mixed evidence for significance of MYC-R + BCL6-R.
	Diffuse large B-cell lymphoma, activated B-cell subtype	ABC-DLBCL	Large cells growing in sheets. CD20+, IRF4+, FOXP1+, BCL6−/+. EBER+ classified separately	CD79B MYD88, CDKN2A, ETV6, PIM1, TBL1XR1, BCL6-R, BCL2-Amp, PRDM1, TNFAIP3, NOTCH2, MYC-R	Multiple subgroups. NOTCH2 / BCL6-R subgroup may be related to marginal zone.	High
	Primary mediastinal large B-cell lymphoma	PMBL	Large cells in sheets. CD20+, CD30+, CD23+, MAL+, c-Rel nuc+	CIITA-R, CD274/PDCD1LG2-R/Amp, REL-Amp, SOCS1, STAT6, PTPN1	Unknown	High	“Gray zone”: features of both PMBL and Hodgkin lymphoma
	Burkitt lymphoma	BL	Blastoid, CD10+, BCL6+, BCL2-neg, simple karyotype with MYC-R	MYC-R, ID3/TCF3, DDX3X, FOXO1, ARID1A, SMARCA4, CCND3, FBXO11	Endemic: EBV-infected B cell w/ concurrent malaria (?)	Very high	Most African endemic and HIV-associated cases are EBER+
T / NK cell
	Adult T-cell leukemia / lymphoma	ATLL	Flower-like nuclei, CD25+, HTLV-1 serology positive, hypercalcemia	PLCG1, PRKCB, CCR4/7, STAT3, TP53, VAV1, NOTCH1, RHOA, CD274-R	HTLV-1-infected T cells, usually since infancy	Low to high	HTLV-endemic regions (Japan, Caribbean, Africa, South America)
	T-cell prolymphocytic leukemia	T-PLL	Circulating medium-sized T cells, CD4+/CD8-neg or double-positive	TRA-TCL1A/B, TRA-MTCP1, JAK3, JAK1, STAT5B	Unknown	High	Increased occurrence in patients with ataxia-telangiectasia
	Angioimmunoblastic T-cell lymphoma	AITL	Follicular dendritic prolif. BCL6+, CD10+, PD1+ T cells. EBV+ B cells	TET2, DNMT3A, IDH2, RHOA, CD28, PLCG1	Abnormal hematopoietic stem cell -> T follicular helper cell	High	T follicular helper phenotype seen in subset of non-ATIL lymphomas
	Peripheral T-cell lymphoma, NOS	PTCL-NOS	Polymorphous, often with eosinophils and histiocytes. Clonal T cells with antigen loss.	TET2/3, DNMT3A, GNA13, RHOA, CD58, FYN, CD28	Unknown	High	Expression of GATA2 versus T-Bet may denote biologically distinct subtypes
	Hepatosplenic T-cell lymphoma	HSTL	Usually TCR gamma-delta+, often CD4-neg and CD8-neg	+7q, STAT5B, STAT3, SETD2, INO80, ARID1B, SMARCA2, TET2/3, DNMT3A	Unknown	High	Predisposed by immunosupression due to solid organ transplant
	Anaplastic large cell lymphoma, ALK+	ALK+ ALCL	Large cells with sinusoidal growth. ALK+, CD30+, cytoxic granule+	NPM1-ALK or other ALK-R	Unknown	High	Variants with atypical morphology (small cell, etc) are ALK+
	Anaplastic large cell lymphoma, ALK-neg	ALK-negative ALCL	Classic ALCL features, CD30+, ALKneg, cytoxic granule protein+	DUSP22/IRF4-R, TP63-R, ROS1-R, TYK2R, FRK-R. JAK1, STAT3, PRDM1.	Unknown	High	Significantly better prognosis in DUSP22/IRF4-R vs TP63-R
	Extranodal NK/T cell lymphoma, nasal type	ENKTL	Angiocentric & angiodestructive. Cytoplasmic CD3+, CD56+, EBER+	DDX3X, TP53, KMT2D, STAT3	EBV-infected NK or T cell	High	Most common in Asian populations.

Note: “Genomic aberrations” lists symbols of genes involved in gene fusion or regulatory element rearrangements (“-R” or partner loci linked by hypen), amplifications (“-Amp”) or mutation / inactivation (no suffix).

Like DLBCL, peripheral T cell lymphoma - not otherwise specified (PTCL-NOS) is a heterogeneous category of biologically diverse aggressive lymphomas. The T-cell transcription factors GATA-3 and T-bet define normal T helper cell subsets (Th2 and Th1), and distinguish two clinically, molecularly, and genetically distinct subtypes of PTCL-NOS^8–10. As in DLBCL, distinguishing these subtypes reliably in a clinical context remains challenging, but could hold significant diagnostic, prognostic, and therapeutic implications^11,12.

The concept of a normal developmental lymphoid stage that is biologically most similar to a given lymphoma subtype has come to be known as “cell of origin” in the lymphoma field. This does not necessarily refer to the cell type in which the neoplastic process began, as lymphomagenesis is a multi-step process. For example, chromosomal rearrangements that lead to activation of the oncogenes BCL2 or CCND1 in B cell lymphomas result from aberrant VDJ recombination in the immature B cell stage¹³. Early oncogenic driver events in chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL/SLL)¹⁴ and hairy cell leukemia¹⁵ can occur in multipotent hematopoietic progenitors, although the fully evolved cancers are most similar to mature memory B cells. Likewise, mutations associated with T-cell lymphomas, including DNMT3A and TET2, commonly occur in the hematopoietic stem cell compartment, and may represent a T cell lymphoma precursor event¹⁶.

Lymphoma grade, corresponding roughly to growth rate, is critical determinant of lymphoma clinical behavior and therapeutic approach. Transformation of low-grade lymphomas to higher-grade lymphomas (typically DLBCL-like) is an ominous clinical event that is often associated with acquisition of additional genetic events, such as inactivation of TP53 or activating MYC rearrangements^17,18.

The lymphoma microenvironment

Relationships between neoplastic lymphoid cells and their microenvironment define the biology of many lymphomas. Early clonal precursors of some lymphomas can be recognized by their immunophenotype in an otherwise architecturally normal lymph node; examples include in situ follicular neoplasia (a precursor of FL)¹⁹ and in situ mantle cell neoplasia, a likely precursor of MCL)²⁰. These clones bear the same oncogene-activating rearrangements as fully evolved lymphomas, but lack the ability to expand beyond the normal microanatomical compartment.

In contrast, fully evolved lymphomas have gained additional genetic hits, and are characterized by their ability to alter the normal architecture of the lymph node or other organ in which they arise²¹. Nonetheless, many well-differentiated lymphomas remain dependent on altered lymphoid structures, such as the expanded follicular dendritic cell meshworks that are essential to the biology of FL and AITL. Recognition of dependency on the follicular microenvironment is critical to the distinction between grade 3 FL and DLBCL, which can be cytologically and immunophenotypically identical. Altered chemokine & chemokine receptor expression, or mutations in genes that regulate lymphocyte trafficking may partially mediate relationships between neoplastic cells and the follicular stroma in germinal center-derived B- can T-cell lymphomas^22,23. Some lymphomas occurring in distinct anatomic sites, such as duodenal-type primary gastrointestinal follicular lymphoma, show dramatically different clinical behavior from genetically and histologically similar systemic lymphomas, potentially due to unique aspects of tumor cell-microenvironment interactions^24,25.

Lymphomas that grow diffusely, such as DLBCL and many PTCL-NOS, are also greatly influenced by microenvironmental factors such as lymphoma-associated macrophages (LAM), blood vessels, fibroblasts and other stromal elements. LAM provide ligands for pathogenic antigen and costimulatory receptors that are expressed by malignant lymphocytes,^21,26. Some lymphomas appear to shape a growth-promoting stromal microenvironment due to gene mutations that result in altered cytokine secretion and stromal cell activation / recruitment^27,28. Gene expression signatures derived from stromal populations are associated with outcomes in several lymphoma types^29,30.

In T-cell lymphomas, gain-of-function mutations in costimulatory receptors^31–33 may potentiate responsiveness of lymphoma cells to ligand presented by LAM and other non-neoplastic cell populations, while deletions of the checkpoint receptor PD-1 may allow evasion of inhibitory microenvironmental signals³⁴. LAM and regulatory T cells may also promote the evasion of host anti-tumor immunity by providing checkpoint ligands and immunoregulatory cytokines^35–38. Recurrent mutations in genes such as TET2 that are associated with clonal hematopoiesis may be present in both T-cell lymphoma cells and in constituents of the microenvironment³⁹, potentially contributing to lymphomagenesis.

Predisposing factors

Genetic predisposition for some mature B-lymphoid cancers, including CLL/SLL and DLBCL, is relatively high compared to common epithelial cancers, based on family registry-based data⁴⁰. Genome-wide association studies (GWAS) have identified genetic polymorphisms linked to increased risk for CLL/SLL , DLBCL, FL and others, but the specific loci identified to date account for only a fraction of the overall apparent familial risk for these cancers⁴⁰. Genetic variants mapping to the HLA loci appear to strongly contribute to risk for several lymphoma subtypes⁴¹. Many lymphoma risk polymorphisms identified in GWAS studies affect non-coding regions with possible roles in gene regulation. For example, a GWAS-identified single-nucleotide polymorphism (SNP) linked to altered risk for CLL/SLL⁴² lies near MYC regulatory elements that have been implicated in CLL/SLL and MCL^43–45, while SNPs linked to DLBCL risk⁴⁶ are located on the opposite side of the MYC gene, in a region containing candidate DLBCL MYC enhancers⁴⁴. Ubiquitous (e.g. EBV) and endemic (e.g. HTLV-1) viruses play direct roles in the development of some lymphomas, while chronic antigenic stimulation due to infection is central to the development of others, such as H. pylori-associated gastric MALT lymphoma⁴⁷. Some lymphomas may be predisposed by complex interactions between genetic, infections, and / or environmental factors. For example, EBV-associated T- and NK-cell lymphomas are more common in populations of Asian and Indigenous American versus European genetic background, despite similar rates of EBV infection⁴⁸. Concurrent EBV and malaria infection have been implicated in the high endemic rate of Burkitt lymphoma in equatorial Africa⁴⁹.

Immune receptors as drivers of lymphomagenesis

Surface immunoglobulin (IG) mediates signaling that is critical for the survival of normal B cells and lymphomas. Utilization of particular IGH gene segments in lymphoma out of proportion to their usage in normal B-cell populations, such as IGHV1–2 or IGHV4 in splenic marginal zone lymphoma⁵⁰, or IGHV3–7 and IGHV3–23⁵¹ in lymphoplasmacytic lymphomas, suggests that recognition of specific antigens (autoantigens or via exposure to specific pathogens) may drive pro-oncogenic signaling in many cases. A phenomenon of cell-autonomous B-cell receptor signaling observed in many cases of CLL/SLL is thought to be mediated by surface IG molecules that are capable of homophilic interactions in the absence of antigen⁵². Similarly, many cases of ABC-DLBCL are dependent on chronic active B cell receptor signaling⁵³ mediated by B-cell receptors with strong autonomous or autoantigen-dependent signaling activity⁵⁴. The somatic hypermutation status of immunoglobulin genes seems to segregate with high- and low-risk forms of both CLL/SLL⁵⁵ and MCL⁵⁶, likely reflecting different natural histories with regard to transit through the germinal center, as well as different co-occurring oncogenic mechanisms^57,58.

Similarly, the T-cell receptor (TCR) mediates signaling that is critical for the survival of normal and malignant T cells. Expression of TCR and downstream scaffold proteins and kinases is maintained in most nodal T-cell lymphomas, with the notable exception of ALK+ ALCL²⁶. For example, the IL-2 inducible T-cell kinase (ITK) regulates the spatiotemporal localization of the TCR signalosome and is a critically important mediator of TCR signaling⁵⁹, and is frequently overexpressed in T-cell lymphomas^60,61. Biased TCR Vβ usage and recurrent gain-of-function alterations in signaling intermediates immediately downstream from the TCR further implicate the TCR in T-cell lymphomagenesis²⁶, and have significant therapeutic implications for these lymphomas¹².

Mechanisms of oncogene dysregulation in lymphoma

B and T lymphocytes employ unique machinery to alter the structure and sequence of antigen receptor genes during normal development, and aberrations in these processes are important contributors to lymphomagenesis. Many recurrent genomic rearrangements seen in mature B and T cell cancers are caused by errors in V(D)J recombination in immature B cells, while other recurrent rearrangements and point mutations are due to off-target activity of activation-induced cytosine deaminase (AID)^62,63, which normally catalyzes IGH class switch recombination and somatic hypermutation in the germinal center. Many rearrangements activate oncogene expression by an “enhancer hijacking” mechanism that involves 3-D looping of distal enhancers present on the partner loci to activate promoters of the involved oncogenes^44,64–67, while a minority of rearrangements encode fusion oncoproteins. Given the variability of genomic breakpoints, particularly for enhancer-hijacking rearrangements, fluorescence in-situ hybridization (FISH) is the most definitive method for their identification in clinical settings, although some rearrangements (IGH-BCL2, IGH-CCND1, ALK) can be inferred based on immunohistochemisty.

Genome-wide evaluation of gene mutations and copy number aberrations has identified fundamental genetic drivers of B cell lymphomagenesis and supported the existence of genetically-determined subtypes of CLL/SLL⁵⁸, MCL⁵⁷, and DLBCL^6,7. More recently, genome-wide CRISPR-Cas9 knockout studies in cell lines have highlighted genes and pathways that are essential for specific lymphoma subtypes^68–70, often corresponding to the same pathways that are altered by recurrent mutations.

Driver mutations: Signaling, motility, and metabolism

Discovery of recurrent mutations in genes encoding cytoplasmic signaling pathways have provided important clues regarding potentially targetable pathways in specific lymphoma subtypes. For example, discovery of the MYD88 L265P mutations in >90% of lymphoplasmacytic lymphoma revealed the central importance of toll-like receptor signaling in that disease⁷¹. Gain-of-function MYD88 mutations are also seen in ABC-DLBCL⁷² and frequently co-occur with mutations of the proximal B cell receptor signaling complex gene CD79B in large cell lymphomas arising in extranodal sites^73–75. Recent work uncovered a novel mechanism of oncogenic signaling via direct physical interactions between the mutant MYD88-containing TLR signaling complex, the BCR/CD79 signaling complex, and the metabolism-regulating mTOR complex within endolysosomal membranes in both DLBCL and LPL⁶⁸. This complex therefore appears to directly regulate both metabolic signaling to mTOR, as well as signaling to NF-kB via the CARD11-BCL10-MALT1 (CBM) axis. The CBM pathway appears to be a unique dependency of ABC-DLBCL as opposed to GCB-DLBCL based on CRISPR screen data⁶⁸, and is the direct target of oncogene-activating rearrangements and tumor suppressor gene deletions in MALT lymphoma⁴⁷. Bruton’s tyrosine kinase (BTK) which mediates signaling from the BCR to the CBM complex can be effectively targeted by small molecule inhibitors such as ibrutinib for the therapy of CLL/SLL and MCL, although a subset of lymphomas may develop resistance due to mutations in BTK or PLCG2, which encodes a direct target of the BTK kinase^76–78.

Genetic alteration of mTOR regulation appears to be central to the biology of FL, which shows recurrent mutations in RRAGC, ATP6V1B2, and ATP6AP1^79,80, genes encoding the amino-acid sensing machinery that controls mTOR activation in response to nutrient availability. ATP6V1B2 mutations appear to decouple mTOR activation from autophagy, potentially sustaining lymphoma cell survival in specific low-nutrient conditions⁸¹. In GCB-DLBCL, the PI3K-AKT-mTOR signaling axis is dysregulated by inactivating mutations of PTEN, amplification of the negative PTEN regulating-MIR17 locus, and change-of-function mutations affecting the downstream transcription factor FOXO1⁸². The PI3-kinase delta inhibitor idelalisib is effective in the treatment of CLL/SLL⁸³, possibly due to the requirement for tonic/autonomous BCR signaling via PI3K in that disease.

Genetic alteration of janus kinase (JAK) signaling is a prominent feature of PMBL (and cHL) in which activity of the pathway is increased by JAK2 gene amplification⁸⁴ and inactivation of the negative regulators SOCS1 and PTPN1⁸⁵. Increased JAK2 signaling is thought to contribute to phosphorylation of STAT6 protein and the STAT target gene activation that is characteristic of the PMBL gene expression program. STAT6 mutations occur in PMBL⁸⁶, cHL⁸⁷, and follicular lymphoma⁸⁸. The function of mutant STAT6 proteins remains unclear, as they show reduced activation-associated phosphorylation⁸⁶, but other evidence points to increased or altered transactivation function^87,88. STAT3 mutations are infrequent but recurrent in GCB-DLBCL^6,89. Apparent loss-of function mutations affecting the STAT3 phosphatase-encoding gene PTPRD appear to be a specific finding in nodal MZL⁹⁰. Mutations in STAT3 and STAT5B are common driver events in mature T cell neoplasms, including T-cell large granulocytic leukemia^91,92 and hepatosplenic gamma-delta T cell lymphoma⁹³. STAT signaling is also activated by ALK fusions and other mutationally activated tyrosine kinases in ALK+ and ALK-negative ALCL⁹⁴.

Gene mutations affecting the MAP kinase pathway are common in several mature B cell cancers. The BRAF V600E mutation is ubiquitous in hairy cell leukemia⁹⁵, which responds clinically to the BRAF V600E-specific inhibitor vemurafenib⁹⁶ while MAP2K1 (MEK1) mutations are common in hairy cell leukemia-variant⁹⁷, and pediatric-type follicular lymphoma⁹⁸. BRAF mutations also occur in a subset of DLBCL⁶.

Mutations also affect signaling pathways related to lymphocyte chemokine response and trafficking, such as CXCR4 in LPL / WM⁹⁹. Genes encoding a set of regulators (S1PR2, P2RY8, GNA13, ARGEF1) that converge on RhoA signaling are recurrently mutated in germinal center B cell lymphomas²², affecting both trafficking and PI3K-AKT signaling. Mutations of the RHOA gene itself are common in AITL^100,101, further highlighting the importance of this pathway in germinal center-derived lymphomas. A gain-of-function mutation in the chemokine recptor gene CCR4 in adult T-cell leukemia/lymphoma (ATLL) impairs receptor internalization and promotes PI3K/AKT signaling upon ligand binding¹⁰².

The diversity of signaling pathway mutations in lymphoma may reflect the degree to which these pathways are “rewired” in different stages of B cell development. Understanding lymphoma subtype-specific signaling dependencies will be critical for prioritizing the growing list of signaling pathway-targeted therapies, and for anticipating and overcoming mechanisms of resistance.

Driver mutations: Epigenetic and transcriptional regulators

Another large category of recurrently mutated genes in lymphoma comprises genes encoding chromatin and epigenetic regulatory proteins. EZH2, which encodes a repressive histone methyltransferase, is targeted by recurrent gain-of-function mutations in GCB-DLBCL and FL^103–105. EZH2 methyltransferase activity is essential for GCB-DLBCL and normal germinal center B cells¹⁰⁶, and specific EZH2 inhibitors have shown promising activity in early phase clinical trials for B-cell lymphoma¹⁰⁷. The histone methyltransferase gene KMT2D and acetyltransferase gene CREBBP undergo recurrent inactivating mutations in several B-cell lymphoma types^89,108, suggesting that the encoded proteins play a tumor suppressor role in lymphoma, possibly by promoting activation of differentiation genes. Recurrent mutations that alter linker histone genes may also affect chromatin regulation in DLBCL¹⁰⁹.

Genetic alteration of DNA methylation pathways have been strongly implicated in AITL, which bears mutations in the DNA methyltransferase DNMT3A and the demethylation pathway gene TET2¹¹⁰, as well as change-of-function mutations in the metabolic enzyme IDH2¹¹¹ that result in oncometabolite-dependent inhibition of DNA and histone demethylation. T-cell lymphomas harboring concurrent TET2 and IDH2 mutations have distinct gene expression and epigenetic profiles, most notably upregulation of TFH-associated genes¹¹². Mouse models of concurrent TET2 and RHOA mutations results in highly penetrant AITL-like lymphomas with strong activation of PI3K and mTOR signaling^113,114.

Other mutations alter transcriptional regulators that act more selectively at DNA sequence-specific targets. In the setting of genomic rearrangements, overexpressed c-Myc (or in rare cases of MCL, N-Myc¹¹⁵) directly binds genes that sustain anabolic cellular metabolism and cell cycle progression¹¹⁶. The transcriptional repressor BCL6 blocks expression of several classes of tumor suppressor genes, including cell cycle inhibitors, DNA damage response genes, and regulators of differentiation¹¹⁷. BCL6 expression is activated in lymphomas by several distinct mechanisms. Activating rearrangements such as IGH-BCL6 occur most commonly in non- germinal center phenotype lymphomas^6,89,118. In contrast, GCB-DLBCL and normal centroblasts show activation of distal BCL6 super-enhancers^119,120, which in turn are bound and activated by the transcription factor MEF2B^44,121. High-level BCL6 expression is also facilitated by non-coding mutations that alter repressive BCL6 protein binding sites in the BCL6 promoter¹²². MEF2B itself is a target of recurrent mutations in B-cell lymphoma that alter DNA-binding properties and interactions with corepressors^121,123,124, although the effect of these mutations on the MEF2B regulome remains poorly understood. Inactivating mutations of PRDM1 (BLIMP1) are common in ABC-DLBCL^6,125, where they likely function to prevent terminal differentiation. Many other developmental transcription factors are recurrently mutated in DLBCL, including EBF1, POU2AF1, POU2F2, IKZF3, IRF4, and IRF86,¹²⁶. Gain-of-function mutations in TCF3 or inactivating mutations of its negative regulator ID3 are genetic hallmarks of BL¹²⁷. Other developmental TF gene aberrations with apparent lymphoma subtype-specific roles include KLF2, which is recurrently inactivated in splenic and nodal MZL^50,90, and FOXP1-activating rearrangements in MALT lymphoma and extranodal DLBCL^128,129.

NOTCH1 and NOTCH2, which encode proteins that serve as both cell-surface receptors and transcription factors, undergo truncating PEST domain mutations in several small B-cell lymphoma types (CLL¹³⁰, MCL¹³¹, MZL^90,132,133, and FL¹³⁴) that increase the stability of the active intracellular form of the protein. Similar gain-of-function Notch gene mutations are enriched in recently described genetic subtypes of DLBCL^6,7, often in concert with BCL6 rearrangements. Since PEST mutations alone do not appear to initiate Notch signaling, interactions with Notch ligand-expressing stromal cells might be critical for Notch-dependent lymphomas, a concept supported by immunohistochemical detection of active Notch in most CLL lymph node biopsies¹³⁵, although active Notch can also be seen in some circulating CLL cells⁴⁵. Intracellular Notch sustains the growth of some MCL cell lines by binding and activating B cell-specific MYC enhancers, and several lines of evidence point to a similar role for Notch in CLL^43,45,136. Whether inhibitors of Notch signaling might play a role in B-cell lymphoma therapy remains to be determined.

The NF-kB family of transcriptional regulators are known to play a critical role in ABC-DLBCL as major downstream effectors of BCR signaling via BTK and the CBM complex^53,137 This canonical pathway of BCR signaling to NF-kB seems to be essential for a subset of MCL, but is dispensable for MCL with mutations in the alternative NF-kB pathway, which may confer resistance to BCR signaling inhibitors⁷⁸. Focal amplification of the NF-kB gene REL is thought to contribute to NF-kB activation in PMBL and cHL^138,139. While the canonical pathway of BCR signaling to NF-kB is inactive in GCB-DLBCL, this disease also shows recurrent focal REL amplifications¹³⁹ and inactivating mutations of the NF-kB inhibitor genes NFKBIA and NFKBIE⁶, suggesting that NF-kB activation by alternate mechanisms may play a role in a subset of GCB-DLBCL.

Driver mutations: DNA damage response, cell cycle regulation, and survival

Mutations or genomic deletions affecting genes that regulate DNA damage response are critical in several lymphoma subtypes. Inactivation of ATM on 11q (often involving concurrent deletion of BIRC3) is common in mantle cell lymphoma⁵⁷, while ATM and TP53-inactivating lesions are associated with more aggressive behavior in CLL¹⁴⁰. In DLBCL, TP53 mutation is a genetic hallmark of a distinct subgroup characterized by frequent copy number aberrations⁶.

Genetic alteration of cell cycle regulatory genes in lymphoma includes the CCND1 rearrangements seen in most MCL, as well are rarer activating rearrangements of CCND2 or CCND3¹¹⁵. Unlike CCND1, CCND3 is often expressed in B cells in the absence of rearrangement, and gain-of-function point mutations can lead to increased CCND3 protein stability in BL and other high-grade B cell lymphomas¹²⁷. Deletion of the cell cycle inhibitory genes CDKN2A and RB1 are also recurrent in DLBCL⁶.

Low-levels of anti-apoptotic proteins leave normal germinal center B cells highly prone to apoptosis, but this vulnerability is overcome in many FL and GCB-DLBCL by BCL2-activating rearrangements. The small-molecule BCL2 inhibitor venetoclax is effective in CLL/SLL¹⁴¹, which expresses BCL2 in the absence of an activating genetic event, while early clinical evidence suggests efficacy in at least a subset of patients with other lymphoma subtypes, including DLBCL, MCL, and FL¹⁴².

Immunosurveillance and immune evasion in lymphomagenesis

The normal immune system plays an important role in fighting emerging lymphoma clones. Patients treated with immunosuppressive therapy following organ or bone marrow transplantation, or in the setting of an autoimmune disorder, are therefore at increased risk for development of lymphoproliferative disorders. Some lymphoid malignancies arising in these settings may show a therapeutic response to a decrease or cessation of immunosuppressive therapy alone. Escape from immunosurveillance also likely plays a role in the development of lymphoproliferative disorders arising in sites that are physiologically immune-privileged (central nervous system, testes), or pathologically sequestered from the immune system.

Immune evasion may be particularly important in the development of virally-driven lymphoid malignancies, such as B and T/NK cell lymphomas driven by Epstein-Barr virus (EBV) or in adult T cell leukemia / lymphoma driven by human T-lymphotropic virus 1 (HTLV)-1, since certain antigens produced by these viruses can be highly immunogenic. However, it is now clear that even non-virally driven lymphomas occurring in immune-competent persons frequently select for genetic mutations that promote escape from immune surveillance. Genomic deletion of HLA loci is common in DLBCLs occurring in immune-privileged sites¹⁴³. Other DLBCLs show recurrent mutations in immune-interaction genes such as CD83, CD58, and CD70⁸⁹, and these mutations may be particularly enriched in specific genetic subgroups⁶. Mutations in B2M¹⁴⁴, a gene required for self-antigen presentation to T cells via HLA-class 1, and CIITA, a gene required for HLA class 2 expression and antigen presentation, are also recurrent in large B cell lymphomas. Mutations that alter chromatin and transcriptional regulation may also contribute to immune evasion, as gain-of-function mutant EZH2 was recently implicated in suppression of major histocompatibility complex (MHC) gene expression in DLBCL¹⁴⁵, suggesting a mechanism by which EZH2 inhibitor therapy might restore immune-mediated lymphoma cell eradication. Rearrangements and amplifications that increase expression of PDCD1LG2 and / or CD274, the genes encoding the T cell checkpoint ligands PD-L2 and PD-L1, are particularly common in primary mediastinal large B cell lymphoma^84,146–148.

Monoclonal antibodies such as rituximab that direct immune responses to neoplastic cells are already a mainstay of lymphoma therapy¹⁴⁹, and as checkpoint inhibitors and cellular immunotherapies continue to emerge as appealing therapeutic options for patients with advanced lymphoma^150–152, mechanisms of immune escape are likely to be of even greater therapeutic significance in the future.

Summary/Discussion

Basic research continues to provide novel insights regarding the etiology, genetics, and biological diversity of mature lymphoid cancers. As therapeutic options continue to expand, integration of traditional diagnostic approaches and molecular analyses will continue to be essential for optimal lymphoma patient care.

Key Points.

• Clinical classification of lymphomas requires integrated assessment of histology, phenotype, genetics, other laboratory findings, and clinical presentation / history.

• Lymphomas alter their local microenvironment, and may rely on other cell types for contact signals and soluble growth factors required for lymphoma cell homeostasis.

• Lymphomas recapitulate gene expression programs that are characteristic of stages in normal lymphoid development

• Driver genetic alterations in lymphoma include rearrangement of cis-regulatory elements, gene fusions, amplifications, gain-of-function mutations, and inactivating events.

• Mutated pathways include immune receptor signaling, metabolic regulation, transcription and chromatin state, trafficking, DNA damage response, cell cycle regulation, apoptosis, and immunosurveillance.