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Chapter8
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Lymphoma genetics
Giovanni Insuasti- Beltran1, Chad M. McCall2 and Eric D. Hsi
1
Department of Pathology, Wake Forest University School of Medicine, Winston- Salem, NC, USA
2
Carolinas Pathology Group, Charlotte, NC, USA
Introduction, 113
Techniques, 113
Burkitt lymphoma, 115
Diffuse large B- cell lymphoma, not otherwise specified, 116
Mantle cell lymphoma, 118
Follicular lymphoma, 118
Lymphoplasmacytic lymphoma, 119
Introduction
The classification and treatment of lymphoma increasingly
rely on genetic information. Lymphomas were originally
classified by microscopic morphologic features and clinical
outcome alone. Some features, such as the Reed- Sternberg
cell in classic Hodgkin lymphoma or neoplastic follicles in
follicular lymphoma (FL), readily correlate with clinical
behavior. However, despite careful morphologic diagnosis,
many lymphoma subtypes have heterogeneous clinical outcomes. Genetic techniques have allowed for greater insight
into lymphoma biology: we now rely upon cytogenetic data
to classify many lymphomas, and we have incorporated gene
expression data into assays to better prognosticate diffuse
large B- cell lymphoma. The presence of specific mutations
adjusts prognosis, such as TP53 mutations, which confer a
worse prognosis in chronic lymphocytic leukemia and mantle cell lymphoma (MCL). Other genetic data are being used
to direct therapy, such as the presence of both MYC and
BCL2 rearrangements in high- grade B- cell lymphomas
(“double hit”). Recently, next- generation sequencing (NGS)
has allowed for a much broader look at mutations and
expression data in lymphoma, which promises the development of more precise classification and better- targeted therapies in the years to come. In this chapter, we will survey
genetic techniques and the important genetic features of
common non- Hodgkin lymphomas.
Marginal zone lymphoma, 119
Chronic lymphocytic leukemia/small lymphocytic lymphoma, 120
Anaplastic large cell lymphoma, 121
Nodal T- follicular helper (TFH) cell lymphoma, 122
Peripheral T- cell lymphoma, not otherwise specified, 122
Conclusions, 123
Further reading, 123
Techniques
The original genetic studies of lymphoma cells were done
using cytogenetic analysis of metaphase karyotypes.
Karyotypes are made by growing cells in culture, then arresting them in metaphase using microtubule- depolymerizing
agents such as colcemid. The cells are then fixed, placed on
slides, treated with the protease trypsin, and then stained by
Giemsa to produce characteristic chromosome banding patterns. Individual chromosomes and specific abnormalities
can then be identified, but the resolution is limited to
megabase changes in DNA. Changes in chromosome number, many translocations, and some more subtle deletions
and insertions can be identified by karyotype. However, karyotypes are limited to specimens with culturable cells, and
culturing solid tissue samples, such as lymph nodes for lymphoma, is more technically challenging than from liquid
samples (e.g., bone marrow aspirate). Many common abnormalities in lymphomas, such as the 13q deletion seen in
many cases of chronic lymphocytic leukemia, are also not
visible.
Other cytogenetic techniques are required to identify
more subtle chromosomal abnormalities. Fluorescence insitu hybridization (FISH) is the most widely used, where
fluorescently tagged DNA probes are prepared for specific
genomic regions and then hybridized to genomic DNA
within fixed cell nuclei. Interphase cells are most commonly
1
Molecular Hematology, Fifth Edition. Edited by Drew Provan and Hillard M. Lazarus.
© 2024 John Wiley & Sons Ltd. Published 2024 by John Wiley & Sons Ltd.
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113
114 Molecular Hematology
Photographs courtesy of Virginia Thurston, PhD, FACMGG
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used for FISH analysis, which obviates the need for cell culture, but metaphase- arrested cells can be used for more fine
localization of probe signals. Chromosome copy number
changes and genomic deletions and duplications can be
identified by counting probe signals per nucleus, such as
probes for trisomy 12 and deletion 17p11 (TP53) in chronic
lymphocytic leukemia. FISH probes can also be used to identify translocations, where one or both translocation partners
are known. “Break- apart” FISH probe sets are used to identify rearrangements of one genetic locus, regardless of the
translocation partner. These sets use two different fluorescent tags, one upstream and one downstream of the locus
being evaluated. Normal cells have fusion signals with two
fluorescent colors superimposed, while rearranged cells
have a separation of the two colors. For example, a MYC
break- apart FISH probe can be used to screen for MYC rearrangements in Burkitt lymphoma (BL) (Figure 8.1A–B).
“Dual- color, dual- fusion” probe sets are the most specific
FISH method and are used when both translocation partners
are known (Figure8.1C–D). Each translocation partner is
tagged with probes with a specific color; if a translocation is
present, then a fusion signal is generated, while separated
single colors indicate normal chromosomes.
However, FISH requires detailed genetic knowledge
of the regions being investigated and is limited to a small
number of genomic regions per assay. The related techniques
comparative genomic hybridization (CGH), array CGH, and
single- nucleotide polymorphism (SNP) arrays provide a
genome- wide evaluation for genetic abnormalities without
needing to interrogate specific regions. CGH, which is seldom used today, hybridizes a mixture of equal amounts of
tumor DNA and normal control DNA, each labeled with a
different fluorescent dye, to metaphase spreads of normal
cells. The amount of tumor and normal DNA signals on different metaphase chromosomes is used to identify possible
deletions and amplifications. Because traditional CGH has
limited resolution, array CGH and, more commonly, SNP
arrays, have replaced them in clinical assays. Array CGH uses
small (~1- megabase) DNA fragments covering the genome,
which are immobilized in a microarray. Tumor DNA is then
hybridized to these arrays to identify chromosomal copy
number changes at a much higher resolution than possible
with traditional CGH or karyotype. SNP arrays are similar to
array CGH but use hundreds of thousands of small oligonucleotide probes representing SNPs across the genome.
Hybridizing tumor DNA to an SNP array allows for similar
identification of copy number changes, but also provides an
assessment of copy- neutral loss of heterozygosity, where both
copies of a chromosome have the same SNP pattern across a
large region. SNP arrays can also be designed to look for
common “hot spot” single nucleotide mutations in tumors.
Cytogenetic techniques, apart from some SNP arrays,
arenot able to identify specific point mutations in DNA. To
evaluate single genetic loci for the presence of mutations,
(A) (B)
(C) (D)
Figure8.1 FISH probes. Examples of different types
of FISH probes. Break- apart probes are shown (A. Normal
pattern; B. Abnormal pattern), as well as dual- color,
dual- fusion (C. Normal pattern; D. Abnormal pattern).
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Lymphoma genetics 115
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polymerase chain reaction (PCR)- based methods are commonly used. PCR is based on the amplification of specific
small regions of DNA by using oligonucleotide primers
upstream and downstream of a region of interest. Cycles of
primer annealing, DNA polymerization using a heat- stable
DNA polymerase, and high- temperature melting of polymerized double- stranded DNA leads to exponential amplification of the target region. The PCR product can be evaluated
for presence/absence (to identify a translocation event) or
size (to identify insertions or deletions at the target locus) by
gel or capillary electrophoresis. The product can also be
sequenced using Sanger sequencing methods to evaluate for
the presence of point mutations. Two common variations on
classic PCR are reverse transcriptase- PCR (RT- PCR), which
uses RNA as its target and adds a reverse transcriptase step to
convert RNA into cDNA, and quantitative PCR (qPCR or
“real- time” PCR), which uses fluorescent probes and the
generation of signals with each PCR cycle to measure the
quantity of input DNA with specific features.
PCR- based methods, like FISH, are limited to evaluating a
small number of specific regions of DNA. Next- generation
sequencing (NGS, or massively parallel sequencing) methods
have become widely available over the past decade, which
allow for the sequencing of many regions of DNA, including
specific panels of genes, whole exomes, whole transcriptomes,
or even whole genomes. The two most common NGS platforms are Illumina- based platforms, which use DNA immobilized on a flow cell and sequenced using fluorescently
tagged nucleotides, and Ion Torrent- based platforms, which
use DNA immobilized in individual wells on a chip, which
measures changes in pH generated by successful DNA incorporation in ongoing DNA synthesis. NGS methods generate
large amounts of sequence data, which are aligned (matched)
to reference genome data using bioinformatics algorithms.
DNA- based NGS methods, including panels, whole exomes,
and whole genomes, are most effective at identifying point
mutations and small insertions/deletions; translocations may
be difficult to identify if they do not take place in exonic
regions or well-
sequenced DNA. RNA- based NGS methods,
which incorporate a reverse transcription step analogous to
RT- PCR, are more sensitive for detecting translocations that
produce fusion RNA transcripts.
NGS- based tests are becoming more common in lymphoma clinical practice but are not as widely used as the
NGS- based panels that are now standard of care in myelodysplastic syndrome, leukemias, and many solid tumors.
Small panels evaluating genes known to have prognostic significance in chronic lymphocytic leukemia, for example, are
readily available. NGS- based methods are gradually replacing PCR- based methods for evaluating B- cell and T- cell
clonality and for determining the somatic hypermutation
(SHM) status of the IGVH gene in CLL. Larger panels of
genes associated with lymphoma pathogenesis and
prognosis are available in some clinical laboratories but are
not commonly used in everyday clinical practice. Whole
exome sequencing of tumor tissue, particularly when compared with germline DNA from the same patient, is also
clinically available but is much more commonly used in the
research setting to identify novel mutations.
Gene expression data in lymphomas can also be interrogated by molecular methods. Gene expression profiling
(GEP) is similar to array CGH: oligonucleotide probes representing genes of interest are hybridized with fluorescently
labeled tumor mRNA. The amount of mRNA bound to each
probe can be quantified to determine the expression level of
the corresponding gene. This method has been critical for
research studies in many lymphomas, such as defining cell
of origin (COO) subtypes (germinal center B- cell [GCB]
and activated B- cell) in diffuse large B- cell lymphoma
(DLBCL, see later). However, GEP is seldom used in clinical
assays given the instability of mRNA and need for fresh/frozen tissue. Robust methods applicable to formalin- fixed tissues now exist but have not yet been widely adopted. Instead,
data from GEP research studies has been used to develop
immunohistochemistry- based assays as proxies for gene
expression, such as the Hans algorithm in DLBCL, which
uses the results of three immunohistochemical stains
(CD10, BCL6, and MUM1) to classify DLBCL as either
GCB or non- germinal center (non- GCB) types. Briefly, the
first step in the algorithm is to determine the expression of
CD10. CD10+ cases are classified as GCB- type. If negative,
the expression of BCL6 is then determined. CD10- and
BCL6- cases are considered non- GBC. If CD10- /BCL6+,
MUM1 expression is then the determinant of the final classification, with MUM1+ cases being considered non- GCB
and MUM- GCB- type. In research, GEP is now largely
replaced by NGS- based methods (i.e., RNAseq), which
allow for the evaluation of the whole transcriptome.
Burkitt lymphoma
BL is an aggressive mature B- cell lymphoma characterized
by a high- proliferation index and MYC gene rearrangements. Clinically, patients present with rapidly growing
masses. Commonly affected sites are the abdomen,
Waldeyer ring, thyroid gland, ovaries, testis, breast, bones
of the face, and lymph nodes. Three subtypes are recognized: the endemic subtype is highly prevalent in some
regions of Africa, whereas the non- endemic (sporadic)
and the immunodeficiency- associated subtypes are more
evenly distributed worldwide. A significant association
with Epstein–Barr virus (EBV) infection is seen in endemic
(>95%) and immunodeficiency- associated (30–40%) cases.
Morphologically, these tumors show a diffuse growth pattern and are composed of monomorphous mid- sized
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8q24.21 = MYC gene
5ʹ
300 kb 200 kb 100 kb
Exon 1
Exon 2 Exon 3
3ʹ
Endemic BL
14q32.33 = IGH gene
5ʹ
Figure8.2 MYC and IGH breaking points in Burkitt lymphoma. Arrows denote the most common translocation breakpoints seen in
endemic, sporadic, and immunodeficiency- associated (ID) BL.
Cμ Sμ
Sporadic/ID BL
lymphocytes, with round nuclei, clumped and dispersed
chromatin, multiple paracentral nucleoli, moderate basophilic cytoplasm, and squared- off cytoplasmic borders.
Mitotic activity is brisk, as well as apoptosis. Many background macrophages containing apoptotic debris are present resulting in the so- called “starry- sky” pattern.
Molecular testing plays a key role in the diagnosis. The
genetic hallmark of BL is translocation of the MYC protooncogene located at chromosome 8q24 to enhancer elements
of the immunoglobulin genes. The most common partner is
the IGH locus resulting in the t(8;14)(q24;q32) translocation
(80% of cases). Less common partner genes are the kappa
(IGK) locus resulting in the t(2;8)(p11;q24) translocation in
about 15% of cases, and the lambda (IGL) locus resulting in
the t(8;22)(q24;q11) translocation in less than 5% of cases.
Most sporadic cases and those associated with immunodeficiencies show breakpoints of MYC in the IGH::MYC nearby
upstream (5′) or within the first exon or intron, and are typically associated with fusion to the IGH@ isotype switch
regions, located upstream of the respective constant (C)
regions. In contrast, most endemic BL cases show MYC
breakpoints farther upstream, over hundreds of kilobases,
from the promoter region and primarily fuse to the IGH@ J
Sporadic/ID BL
Eμ
H
JJJJJDDDDD VV
Endemic BL
These mutations are higher in sporadic BL (~70%), compared to endemic cases (~40%). Mutations/deletions of
TP53 can also occur in 25–50% of cases, particularly in
EBV- negative ones. EBV- positive cases show higher levels
of activation- induced cytidine deaminase mRNA expression and SHM, particularly in non- coding regions near the
transcription start site.
Finally, GEP shows a unique BL signature, with high- level
expression of MYC and targeted genes, expression of a subset
of GCB genes, and low- level expression of NF- kB target genes
and MHC class I genes. Specifically, based on recurrent copy
number variants (CNVs) and significantly mutated genes,
three distinct clusters have been identified: DGG- BL, IC- BL,
and Q53- BL. DGG- BL cases show predominance ofDDX3X,
GNA13, and GNAI2 mutations, as well as downregulation of
IRF4 and TNFRSF13B. Mutations in ID3 and CCND3, and
overexpression of IRF4 and TNFRSF13B characterize the ICBLL subgroup. The Q53- BL subgroup shows enrichment for
TP53 mutations, but lacks other driver mutations or CNVs.
When patient outcomes among these BL genetic subgroups
are compared, adult BL patients within the Q53- BL have
shown inferior overall survival (OS), while pediatric patients
clustering as DGG- BL had inferior outcomes.
t(2;8)
t(8;22)
3ʹ
region. In IGK::MYC and IGL::MYC, the breakpoints are
usually downstream (3′) of MYC. (Figure8.2) Translocations
of MYC to other non- IG genes are controversial in BL, as
they may represent other high- grade B- cell lymphomas.
Diffuse large B- cell lymphoma, not
otherwise specified
NGS has identified a high frequency of mutations in the
TCF3 gene and silencing mutations in its negative regulator
ID3, leading to activation of the PI3K pathway, and result-
ing in overall increased cell proliferation and survival.
Diffuse large B- cell lymphoma, not otherwise specified
(DLBCL, NOS), is the most common type of non- Hodgkin
lymphoma in the Western hemisphere. Application of
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Lymphoma genetics 117
Cell of origin
Genetic signatur
Associated genes
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Activated B-cell
MCD A53 N1 BN2 EZB
e
C5
MYD88
CD79B
BCL12
Figure8.3 Overview of different DLBCL classification systems including cell of origin, genetic signatures with associated genes.
C2 C1 C0 C3 C4
TP53 NOTCH1
molecular techniques such as GEP and DNA and RNA
sequencing has deepened our understanding of the pathobiology of DLBCL, NOS. This has resulted in an evolving
framework that will likely lead to further refinements in classification and the identification of potential new therapeutic
targets. Despite all these significant advancements, our
understanding of DLBCL remains incomplete due to its
highly heterogeneous nature.
Morphologically, DLBCL, NOS is characterized by partial
or complete effacement of the normal architecture (nodal or
extranodal) by medium to large- sized lymphoid cells with
vesicular mature chromatin. Large size is defined compared
to adjacent macrophage (same size or larger) or residual normal lymphocyte (greater than twice the size) nuclei. Mitotic
activity is generally high, with variable amounts of apoptotic
debris, tingible body macrophages, and necrosis. Other features, such as fibrosis and sclerosis, may also be present.
Traditionally, based on morphologic features alone, three
main cytologic variants have been recognized: centroblastic,
immunoblastic, and anaplastic.
GEP has identified two molecular subtypes based on
resemblance to either GCBs (so-
called GCB subtype) or
activated B- cells (so- called ABC subtype. This has resulted
in the concept of distinction of DLBCL, NOS based on the
COO. GCB cases are associated with a genetic programming
characteristic of germinal center differentiation, including
genes encoding for cell- surface molecules such as CD10
and CD38, nuclear factor A- myb, and the DNA repair
protein8- oxoguanine DNA glycosylase (OGG1), as well as
alterations in BCL6, LMO2, and BCL7A genes. ABC cases are
characterized by constitutive activation of the B- cell receptor
(BCR) signaling and NF- kB pathways, with alterations associated with a post- germinal center environment, including
IRF4, BLC2, and FLIP (CFLAR) genes.
Unclassied Germinal center B-cell
BCL6
NOTCH2
EZH2
PTEN
BCL2
KMT2D
Recently, several studies have revealed a highly heterogeneous molecular landscape with a relatively high frequency of
mutations, somatic copy number alterations (CNAs), and
structural variants (SVs), averaging 7.8 driver mutations per
case, with the total number of mutations typically higher in
GCB COO than in ABC COO. Using NGS or a combination
of GEP and NGS, the complexity of DLBLCs has been further
highlighted. Based on this data, different genetic subtypes
have been reported. The MCD/C5 subgroup was characterized by MYD88 L265P and CD79B mutations, the N1 group
showed NOTCH1 mutations, the BN2/C1 cluster showed a
predominance of BCL6 translocations and NOTCH2 mutations, and the A53 subgroup associated with TP53 inactivation and aneuploidies. All these subtypes correlate with the
ABC COO. Subtypes associated with the GCB COO phenotype are the EZB/C3 (EZH2 mutations and BCL2 translocations) and ST2 (SGK1 and TET2 mutations) (Figure8.3)
Classifications based on genomic signatures provide a way
to rationalize the heterogeneity of these tumors into subtypes
that share a common biological pathogenesis and, therefore,
may respond similarly to targeted therapies. Unfortunately,
despite the overall similarities among signature clusters generated to date, no unified concept for consensus clusters and
their significant genetic drivers has been officially established, precluding the definition of a single unified genetic
framework for DLBCLs.
While the currently approved regimens are prescribed
regardless of COO, the differences in signaling pathways
involved in the pathogenesis of each subtype likely explain
differences in responses to therapies that also include targeted
agents. Early evidence of this difference has been observed in
trials of targeted agents such as REMoDL- B, which in longterm follow up demonstrated improved outcomes for patients
of ABC subtype treated with bortezomib plus R- CHOP.
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Mantle cell lymphoma
MCL represents about 3–10% of B- cell lymphomas, affecting
predominantly elderly patients (median age ~68 years).
Clinically, most cases present with peripheral lymphadenopathy, splenomegaly, and bone marrow involvement.
Extranodal involvement may also be seen, such as in the gastrointestinal tract (classically seen as lymphomatoid polyposis) and Waldeyer ring. At the morphologic level, these
lymphomas are characterized by diffuse, nodular growth, or
mantle zone patterns, by a proliferation composed of small
cells with slightly irregular nuclear contours, mature chromatin with inconspicuous nucleoli, and scant cytoplasm.
More aggressive forms of MCL, like the pleomorphic and
blastoid variants, usually show dispersed or blastic chromatin, increased mitotic activity, and proliferative indices. In
general, MCL is considered an aggressive disease, although a
more indolent, less common, form designated as nonMCL (nnMCL) has also been described. nnMCL patients
present with splenomegaly and peripheral blood involvement without lymphadenopathy. Phenotypically, MCL cells
usually show strong expression of B- cell- associated markers
(CD19, CD20, CD79a, and PAX5), surface light chain restriction, and co- expression of CD5, cyclin D1, CD43, and FMC7.
They are usually negative for CD10, BCL6, and CD23.
The t(11;14)(q13;q32) CCND1::IGH translocation is the
genetic hallmark of MCL, present in more than 95% of cases.
Occasionally, light chain loci (IGK or IGL) serve as the translocation partner. Due to the translocation, overexpression of
the cell cycle regulatory protein cyclin D1 activates cyclindependent kinases (CDKs), which overcome the regulatory
effects of RB1 and p27, critical during the G1- to- S phase
transition. Therefore, overexpression of cyclin D1 overcomes
the late G1 phase checkpoint, leading to increased cell proliferation, survival, and malignant transformation. A small
subset of cases are negative for CCND1 rearrangements.
Dysregulation of other cyclin family genes (CCND2, CCND3,
CCNE1, or CCNE2) has been identified among those cases.
Given the fact that most of the MCL cases derive from naïvelike B- cells, the majority show unmutated immunoglobulin
heavy chain gene variable (IGHV) genes and typically high
expression of the SRY- box transcription factor 11 (SOX11)
levels. A minority of cases, including nnMCL, derive from
memory- like B- cells, showing a higher rate of SHM and
decreased levels of SOX11.
Secondary genomic alterations, particularly gains or amplifications of 3q, 7p, 8q (MYC), 15q, 18q (BCL2), and losses of
1p, 2q, 6q, 8p, 11q (ATM), 13q (RB1), 17p (TP53) and 19p,
are frequently identified. NGS has identified a complex
molecular landscape. Frequent mutations include ATM (most
common, 40–60%), TP53, 3′ untranslated region (3′- UTR)
of CCND1, KMT2D, and BIRC3. Less- common mutations
nodal
occur in NSD2, NOTCH1/2, CARD11, SMARCA4, SP140,
among others. The most common alterations in nnMCL are
in CCND1 (5′- region) and TP53. TP53 alterations are associated with poor prognosis in patients undergoing chemotherapy and autologous hematopoietic cell transplantation.
Post- hoc analyses of trials of chemoimmunotherapy and
hematopoietic cell transplant in MCL showed that younger
patients with del(17p) or mutations of TP53 derive little
orno benefit from chemoimmunotherapy and autologous
hematopoietic cell transplantation. The recent results of the
TRIANGLE trial demonstrated benefit of the addition of
the BTK inhibitor ibrutinib to induction chemoimmunotherapy and subsequent maintenance (with or without
transplant), providing an option for the treatment of this
patient population who have a generally poor prognosis.
This treatment strategy has recently been adopted by the
NCCN guidelines for the treatment of B- cell malignancies.
Follicular lymphoma
FL is a mature, indolent B- cell lymphoma. It accounts for
10–20% of all lymphomas. Most patients present with widespread disease at diagnosis, including significant lymphadenopathy and splenomegaly. The bone marrow is affected in
40–70% of cases. The cells of origin are germinal centerderived (GC) B- cells. Morphologically, classic FL shows a
predominantly follicular growth pattern, with a proliferation
composed of small, cleaved cells with mature chromatin and
scant cytoplasm (centrocytes), and admixed larger cells with
round nuclei, vesicular chromatin, and multiple peripherally
origin, in addition to strong expression of B- cell- associated
markers (CD19, CD20, CD79a, PAX5), the neoplastic cells
usually co- express BCL2 and germinal center- associated
markers (CD10, BCL6, LMO2, MEF2B, GCET1, GCET2) in
the classic variant of FL.
The characteristic t(14;18)(q32;q21) translocation is present in about 85–90% of FL. This translocation juxtaposes
the antito constitutive BCL2 overexpression. More than 90% of FL
have associated CNAs, including gains of chromosomes 1q,
2p, 7. 18 and X, and deletions of 1p and 6q. Common gene
mutations occur over time and involve (1) recurrent mutations in histone modifier genes, particularly EZH2. EZH2 is
a key component of the polycomb repressive complex 2
(PRC2). These mutations often result in gain- of- function
alterations, leading to increased trimethylation of histone
H3lysine 27 (H3K27me3) and enhanced repression of target
genes; (2) DNA methylation regulators including CREBBP
and KMT2D; (3) BCR signaling, with recurrent mutations
identified in genes involved in this pathway, such as CD79B
and CARD11; and (4) nuclear factor- κB (NF- κB) pathway,
apoptotic gene BCL2 next to the IGH locus, leading
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Lymphoma genetics 119
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including key components such as MYD88 and TNFAIP3
(A20), leading to constitutive activation of NF- κB signaling,
promoting cell survival and proliferation. Mutations in other
important genes like TNFRSF14 are important to foster a
supporting tumor microenvironment. None of these pathways, however, have been specifically associated with an
increased risk of transformation, as the sequences of events
driving disease progression remain largely unknown. BCL6
rearrangements can be seen in 15–20% of BCL2- rearranged
FL but is higher (about 35%) in cases lacking BCL2 rearrangements. Finally, cases lacking BCL2 and BCL6 rearrangements (10–15% of classic FL) show evidence of SHM
with GEP reminiscent of late/post- GC cells. The frequency
of mutated genes varies compared to BCL2- rearranged counterparts, with STAT6 and KMT2D being the most frequently
identified.
Classic FL is considered an indolent process, with longterm survival (>15 years) with current therapies. The FLInternational Prognostic Index (FLIPI) has prognostic
significance on outcomes, with a modification available (m7FLIPI) which includes the molecular status of seven genes
(EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and
CARD11). EZH2 mutations have been associated with
favorable prognosis in patients treated with immunotherapies and are predictive of response to approved EZH2 inhibitors. Identifying molecular predictors of early relapse or
high- grade transformation, as well as better stratification
factors in the context of a quickly evolving therapeutic landscape remain as active investigational areas.
Lymphoplasmacytic lymphoma
Lymphoplasmacytic lymphoma (LPL) is an indolent mature
B- cell lymphoma composed of a spectrum of small lymphocytes, plasmacytoid lymphocytes, and plasma cells. It represents about 2% of all hematological malignancies and is most
prevalent in elderly (>60 years), white and male populations.
LPL usually involves the bone marrow, and sometimes
lymph nodes and spleen. Bone marrow involvement with an
IgM monoclonal paraprotein in blood defines Waldenstrom
Macroglobulinemia (WM).
LPL is primarily associated with a singlein the MYD88 gene (L265P). This mutation occurs in
93–97% of cases, and it is considered a hallmark genetic
alteration. MYD88 is an adapter protein involved in the Tolllike receptor (TLR) signaling pathway, which plays a critical
role in immune response regulation. The MYD88 L265P
mutation leads to constitutive activation of the TLR signaling pathway, resulting in increased cellular proliferation and
survival, through activation of nuclear factor- kappa B (NF-κB),
a transcription factor that regulates gene expression in
cellgrowth, inflammation, and apoptosis. The second most
point mutation
common genetic alterations observed in about 30–40% of
LPL/WM are somatic mutations in the C- terminal region of
the CXCR4 gene. More than 40nonsense and frameshift var-
iants have been described. CXCR4 functions as a chemokine
receptor that regulates lymphocyte trafficking and homing.
Mutations lead to impaired internalization and degradation
of CXCR4, resulting in enhanced chemotaxis and retention
of lymphocytes within the lymphoid tissue. This abnormal
lymphocyte trafficking pattern contributes to the accumulation of lymphoplasmacytic cells and the development of
LPL. CXCR4 mutations have been associated with high IgM
serum levels, symptomatic hyperviscosity, shorter time- totreatment initiation, and lower response activity to ibrutinib
therapy. About 50% of LPL/WM patients have a heterozygous loss of 6q, affecting important regulatory genes like
IBTK, BCLAF1, HIVEP2, TNFAIP3, and FOXO3. Loss of 6q
is mutually exclusive of CXCR4 mutations. Rare mutations in
TP53 have been reported in a subset of LPL cases and are
associated with an aggressive clinical course.
Marginal zone lymphoma
Marginal zone lymphoma (MZL) represents a heterogeneous
group of B- cell malignancies arising from marginal zone
B- cells. Extranodal MZL of mucosa- associated lymphoid tissue (EMZL), nodal marginal zone lymphoma (NMZL), and
splenic marginal zone lymphoma (SMZL) are recognized as
distinct entities. Despite having distinct etiologies and
genetic changes, these lymphomas have overlapping histologic and immunophenotypic features. Similarly, they share
common dysregulation of key signaling pathways important
to the homeostasis of normal marginal zone B- cells, such as
BcR, NF- κB, and NOTCH.
EMZL accounts for approximately 10–20% of all lymphomas. Symptomatology depends on the affected site. They
usually present as localized lesions, and some may be discovered incidentally. These lymphomas affect most commonly
the stomach, ocular adnexa, salivary glands, skin, lung, breast,
and thyroid, but any site, including sites lacking mucosa,
canbe involved. In general, they are often associated with
anunderlying chronic inflammatory process. Infections by
Helicobacter pylori (stomach), Campylobacter jejuni (small
intestine), Borrelia burgdorferi (cutaneous), and Chlamydia
psittaci have been commonly associated etiologic factors.
Autoimmune conditions like Sjögren syndrome and
Hashimoto thyroiditis increase the risk of salivary and
thyroid EMZLs, respectively.
At the genetic level, alterations vary according to the
anatomical site. The t(11;18)(q21;q21) BIRC3::MALT1 trans-
location is seen most often in gastric (24%) and pulmonary
(40%) MALT lymphomas. This translocation causes
canonical and non- canonical activation of NF- κB pathways.
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Its presence in H. pylori- positive gastric EMZL has been
associated with a lack of response to antibiotic treatment.
Other common translocations include the t(14;18)(q32;q21)
IGH::MALT1 usually found in lung and ocular adnexa
EMZL; t(3;14)(p14.1;q32) FOXP1::IGH associated with thyroid and ocular adnexa EMZL and primary cutaneous cases;
t(1;14)(p22;q32) BCL10::IGH is most frequently present in
gastric and lung lymphomas. Mutations in genes involved in
the NF- κB signaling pathway, such as TNFAIP3 have been
reported in all types of EMZLs, but particularly overrepresented in ocular adnexal lymphomas.
Splenic and nodal MZLs have a different genetic composition. About 30% of splenic MZLs show hemizygous deletion
of 7q31- 32, and more infrequently, they harbor recurrent
translocations juxtaposing the CDK6 oncogene to immuno-
globulin gene loci. Molecularly, two genetic clusters have
been described in splenic MZLs (SMZL). The first is characterized by mutations affecting NF- κB (e.g. TNFAIP3),
NOTCH family (NOTCH1, NOTCH2, SPEN), and KLF2,
termed NNK cluster (~60% of cases). The second cluster
shows mutations in DNA repair genes (e.g. TP53, ATM),
MAPK, and TLR (e.g. MYD88) signaling genes, called DMT
cluster (~30% of cases). Interestingly, NNK- SMZLs are
enriched in IGHV1- 2*04 usage and 7q31- 32 deletions, while
DMT- SMZLs lack both features. Nodal MZLs also share a
common mutational background in NOTCH family genes
(NOTCH1, NOTCH2, and SPEN) with SMZLs. On the other
hand, BRAF and PTPRD mutations are nearly exclusive to
nodal MZLs. Finally, trisomies of chromosomes 3 and 18 are
observed in 25% of all subtypes of MZLs. These cytogenetic
and/or molecular features can aid in the differentiation of
MZLs from other low- grade B- cell lymphomas.
Chronic lymphocytic leukemia/small
lymphocytic lymphoma
Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a mature B- cell lymphoma composed of
monomorphic small lymphocytes that frequently co- express
CD5 and CD23in addition to B- cell- associated antigens. For
diagnostic purposes, CLL requires a neoplastic cell count of
at least 5 × 10*9/L in peripheral blood, with characteristic
morphology and immunophenotype. In tissues, SLL is diagnosed when organ enlargement due to neoplastic infiltration
is present (including lymph nodes). At the cytogenetic and
molecular level, CLL/SLL is a very heterogeneous disease.
SHM of the IGHV region plays an important prognostic
role in CLL/SLL. The presence or absence of IGHV mutations is a strong prognostic factor and has significant implications for disease progression and treatment response.
Mutated CLL (M- CLL) cases are characterized by a higher
number of mutations in the IGHV gene compared to the
germline sequence (i.e. <98% identity). These mutations are
indicative of SHM and suggest that the CLL clone originates
from a postwith a more indolent clinical course, longer time to treatment, and better overall survival compared to unmutated
CLL (U- CLL) cases. In contrast, U- CLL lacks significant
mutations in the IGHV gene, resembling germline configuration (i.e. >98% identity). U- CLL is associated with a more
aggressive disease course, shorter time to treatment, and
inferior outcomes. The absence of SHM in U- CLL suggests
that the disease may originate from pre- germinal center B
cells or memory B cells that have undergone limited or no
SHM. The different mutational status of the IGHV gene
reflects distinct cellular origins and likely contributes to differences in disease biology and response to therapy. M- CLL
cells generally exhibit a more mature phenotype, while
U- CLL cells often display features associated with less
mature B cells. Detailed immunogenetic analysis based on
similarities in the amino acid patterns of the BCR led to the
concept of stereotypy, with 29major stereotypes identified
in CLL/SLL. Some of these, like the IGHV3- 21/IGLV3- 21,
have been associated with poor prognosis, regardless of
SHM status. Point mutations, particularly the IGLV3- 21
R110, may also be associated with inferior outcomes. These
differences may influence cellular signaling, proliferation
rates, and responses to microenvironmental stimuli.
Genetically, the most frequent chromosomal alterations
in CLL/SLL are deletions of 13q (50–60%; DLEU2- mir-
15- 16 cluster), 11q (10–20%; ATM gene), 17p (5–10%; TP53
gene), and trisomy 12 (15–20%). Of these, deletion of TP53
confers a worse prognosis and predicts suboptimal responses
to chemotherapy. Additionally, the presence of complex
karyotypes (≥5 abnormalities) is also associated with unfavorable outcomes. The best prognostic group includes
patients having deletion 13q as a sole abnormality. The presence of 11q and 17p deletions have been associated with
more extensive lymphadenopathy, splenomegaly, cytopenias, and B symptoms.
While no diseaseNGS has unveiled mutations affecting different pathways
and cellular programs. The most frequently mutated genes
in treatment naïve CLL/SLL are NOTCH1 (10–15%), ATM
(10–15%), SF3B1 (10%), TP53 (5–10%), and BIRC3 (5%).
The frequency and composition of chromosomal abnormalities and mutations vary according to IGHV gene SHM
status. For instance, patients with M- CLL commonly carry
deletion 13q or trisomy 12, and/or mutations in MYD88 and
CDH2, whereas those with U- CLL status show deletions
11q, 17p and/or TP53, ATM, SF3B1, and NOTCH1 muta-
tions. In relapsed cases, the frequency and type of mutations
may differ, with alterations in TP53 (20–30%), MYC gain
(15%), and CDKN2A losses (10%) often seen, with TP53
mutations particularly contributing to worse outcomes.
germinal center B cell. M- CLL is associated
defining mutations exist in CLL/SLL,
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Finally, mutations in BTK (mainly in the kinase domain),
PLCG2, and CARD11 can be identified in patients treated
with BTK- inhibitors, leading to disease progression.
Similarly, mutations in the BCL2 gene affecting the drugbinding site may be present in patients under therapy with
BCL2- inhibitors. Testing for these mutations may be beneficial in recurrent/relapsed disease.
Table8.1 summarizes the most important genetic altera-
tions in B- cell lymphomas.
Table8.1 Summary ofmost important genetic alterations inB- cell lymphomas
Important cytogenetic/
Entity
Burkitt lymphoma MYC rearrangements:
Diffuse large B- cell
lymphoma, NOS
Mantle cell lymphoma • CCND1 rearrangement:
Follicular lymphoma BCL2 rearrangement: t(14;18)
Lymphoplasmacytic
lymphoma
Extranodal marginal zone
lymphoma of
mucosa- associated
lymphoid tissue (EMZL)
Splenic MZL • del(7q), +3, +18
Nodal MZL • +3, +18
Chronic lymphocytic
leukemia/Small
lymphocytic lymphoma
(CLL/SLL)
Molecular findings
•
t(8;14) MYC::IGH (80%)
• t(2;8) IGK::MYC (15%)
• t(8;22) MYC::IGL (5%)
• Multiple gene mutations
andCNVs
• COO determination (GC vs
ABC subtypes)
t(11;14) CCND1::IGH
• Less common: CCND2/CCND3
rearrangements
• TP53 mutations
IGH::BCL2
• MYD88 L265P
• CXCR4 mutations
MALT1, BCL10, FOXP1
rearrangements:
• t(14;18)(q32;q21) IGH::MALT1
• t(3;14)(p14.1;q32) FOXP1::IGH
• t(1;14)(p22;q32) BCL10::IGH
+3, +18
• KLF2, NOTCH2 mutations
• KLF2, NOTCH2, PTPRP
mutations
• IGHV mutation status
• del(11q), +12, del(13q),
del(17p)
• TP53mutations
• Detection of complex
karyotypes (≥ 5 abnormalities)
Anaplastic large cell lymphoma
Anaplastic large cell lymphomas (ALCL) are mature T- cell
lymphomas characterized by large neoplastic lymphoid cells
with pleomorphic horseshoe- shaped or reniform nuclei and
ample cytoplasm and strong and uniform expression of
CD30. Two main subtypes are identified based on the expression of the anaplastic lymphoma kinase (ALK) protein: ALKpositive and ALK- negative ALCLs.
Testing
methodology Other
FISH
Cytogenetics
IHC
FISH (rule out MYC,
BCL2 and/or BCL6
rearrangements)
FISH
IHC/HTS (for TP53)
FISH
Cytogenetics
AS- PCR
HTS
FISH
Cytogenetics
FISH
Cytogenetics
HTS
FISH
Cytogenetics
HTS
Sanger sequencing
FISH
Cytogenetics
HTS
GEP subtypes described
GEP subtypes described
GEP for proliferation and
signatures of nnMCL vs cMCL
EZH2 mutation detection may be
indicated in relapsed/refractory
disease
R110
BCR stereotyping and IGLV3- 21
mutation status may be important
for risk stratification
BTK, PLCG2 and BCL2 mutations for
therapy resistance
Abbreviations: FISH=fluorescence in- situ hybridization; GEP=Gene expression profiling; CNVs=copy number variants; COO=cell of origin;
IHC=immunohistochemistry; HTS = high- throughput sequencing; AS- PCR=allele- specific polymerase chain reaction.
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ALK- positive ALCL is a rare entity, comprising about 3%
of adults and 10–15% of pediatric non- Hodgkin lymphomas. Nodal and extranodal involvements are frequent. At
the molecular level, these lymphomas are characterized by
ALK overexpression secondary to chromosomal rearrangements that juxtapose the 3′ portion of the ALK gene on
chromosome 2p23 to the 5′ portion of a given partner gene
that promotes constitutive expression and activation of the
kinase function. The most common fusion partner is NPM1
located on 5q35, resulting in the t(2;5)(p23;q35) NPM1::ALK
fusion transcript. Over 20 additional gene partners have
also been identified. Overexpression of ALK leads to activation of numerous cellular signaling pathways including
JAK/STAT2, STAT5, PI3K/AKT, RAS/ERK/MAPK, among
others. The prognosis of ALK- positive ALCL patients is
good, with long- term survival rates approaching 80%. The
availability of targeted therapies, such as ALK- inhibitors
may improve prognosis.
ALK- negative ALCL represents about 5–15% of mature
T- cell lymphomas, most occurring in adults (~54 years).
Compared to ALK- positive ALCL, they show a more heterogeneous molecular landscape. About 20–30% of cases
show rearrangements of the dual specificity phosphatase
22 (DUSP22) locus at 6p25.3. These cases show DNA
hypomethylation leading to increased immunogenicity
through decreased expression of PD- L1 (programmed
death ligand 1) (programmed death ligand 1), and overexpression of CD58 and HLA class II molecules. Activating
mutations of JAK1 and STAT3 (~30%), rearrangements of
TP63 (~5%), and loss of 17p13 (TP53) have also been identified. Clonal rearrangements of T- cell receptor genes are
identified in most cases. The prognosis of ALK- negative
ALCL is worse than ALK- positive cases, with poor outcomes seen particularly among TP63 rearranged cases or
cases showing loss of TP53.
Nodal T- follicular helper (TFH) cell
lymphoma
Nodal TFH cell lymphomas (nTFHLs) are mature T- cell
lymphomas derived from T- follicular helper cells. They are
one of the most common non- cutaneous T- cell lymphomas,
accounting for 12–35% of cases. Most nTFHL cases are
nTHFL- angioimmunoblastic type (nTFHL- AI, previously
termed angioimmunoblastic T- cell lymphoma), with two
other rare subtypes (nTHFL- follicular type and nTHFLNOS). They express at least two TFH phenotypic markers,
with the most common clinically available markers being
BCL6, CD10, PD1 (CD279), ICOS, and CXCL13. nTFHLs
are also usually positive for CD4. The hallmark of nTFHL- AI
is the proliferation of high endothelial venules and follicular
dendritic cells (FDCs) outside of lymphoid follicles, and
EBV- positive B- cells are often present in the background.
Many nTHFL- AI cases contain clonal B- cell populations,
which may progress to diffuse large B- cell lymphomas.
nTHFL, follicular type, has a follicular growth pattern without
the extrafollicular vascular and FDC proliferation of AI type.
nTHFL- NOS is limited to unusual cases that do not meet
diagnostic criteria for the other two categories.
nTFHLs have a unique pattern of genetic mutations, best
characterized in the angioimmunoblastic type. Up to 80%
of nTHFL- AI have inactivating mutations in TET2 and up
to 70% have dominant negative inactivating G17V point
mutations in RHOA. Other common mutations include
loss of function mutations in DNMT3A and gain of function mutations in IDH2. RHOA G17V and IDH2 R172muta-
tions are only rarely detected in non- TFH peripheral T- cell
lymphomas, and their presence should suggest an nTFHL
diagnosis. Mutations in components of T- cell receptor
signaling pathways, such as PLCG1, CD28, FYN, and VAV ,
may also be detected in nTFHLs. The DNMT3A and TET2
mutations in nTFHLs can also be detected in admixed B
cells and hematopoietic stem cells of nTFHL patients, suggesting an underlying stem cell mutation preceding nTFHL
development. In addition to point mutations, the t(5;9)
(q33;q22) ITK::SYK translocation has been reported in 20%
of nTFHL- follicular type cases, and is occasionally detected
in other nTFHL subtypes.
Peripheral T- cell lymphoma, not
otherwise specified
Peripheral T- cell lymphomas, not otherwise specified
(PTCL- NOS), are nodal and extranodal T- cell lymphomas
that do not meet criteria for specific T- cell lymphoma entities. 20–35% of T- cell lymphoma cases fall into this category,
which is associated with aggressive clinical behavior and
poor response to therapy. Patients usually present with lymphadenopathy and constitutional symptoms; lymph nodes
are most commonly involved, but extranodal disease can be
seen. PTCLtures, most often containing medium to large, pleomorphic
neoplastic cells, and often containing a mixed inflammatory
cell background. They are most often CD4- positive, and frequently show loss of one or more pan- T- cell surface antigens,
such as CD5, CD7, or CD2. Cytotoxic T- cells markers are
seen in a minority of cases.
Although PTCL- NOS cases are heterogeneous, two
molecular subtypes have been characterized by gene expression analysis: PTCL- GATA3 and PTCL- TBX21, which overexpress the transcription factors GATA3 (associated with
Th2 T- cell differentiation) or TBX21 (Th1 differentiation),
respectively. PTCL- GATA3 lymphomas are associated with
poorer prognosis and have more genomic complexity than
NOS cases have heterogeneous microscopic fea-
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