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Oquendo et al. J Transl Genet Genom 2021;5:89-111 https://dx.doi.org/10.20517/jtgg.2021.04 Page 97

[73]
transcriptional regulation [Figure 3]. Shanmugam et al. propose that in the case of SMZL, NOTCH2
mutations are not initiating events, but that the sustained signalling provided by these mutations provides a
selective advantage in tumours that have already established themselves in a ligand-rich microenvironment.
This is supported by evidence in mice where those with activating Notch2 mutations in mature B-cells
displayed expansion of the marginal zone but did not develop lymphoma . Additionally, NOTCH2
[74]
[73]
mutated tumours exhibit sub-clonal heterogeneity, with consequent aggressive clones , and inferior
survival that has been demonstrated in clinical studies [24,54] . Expression of the Notch intracellular domain 2
(NICD2) can be detected in SMZL cases and is a common feature of both NOTCH2 wild-type and mutated
SMZLs , similar to prior findings with NOTCH1 in CLL , suggesting that Notch activation is a general
[75]
[73]
feature of SMZL tumour cells. The work by Shanmugam et al. showed higher frequency of NICD2+ cells
[73]
in mutated versus wild-type tumours and higher in the marginal zones of the white pulp. It is yet to be
determined if enhanced NICD2 expression in wild-type tumours is explained fully by mutations in other
Notch regulators, such as NOTCH1 (~5%) and SPEN (~5%) [24,57] , structural or copy number aberrations, or
by the enrichment of NOTCH2 in the normal counterpart of SMZL.

TP53 mutations account for up to 15% of SMZL
TP53 is one of the main SMZL associated genes implicated in cell cycle control, along with CCND3 (~5%)
and ATM (~4%). However, the lack of germline material in many of the studies makes it difficult to confirm
whether ATM mutations are truly somatic and should be looked at with caution. TP53 is disrupted in 10%-
15% of SMZL cases [24,57] . Most TP53 mutations are missense mutations within the DNA binding domain
[Figure 3], attenuating or eliminating its function as tumour suppressor, since mutant proteins lose the
ability to activate canonical p53 target genes. This leads to uncontrolled cell proliferation and permissive
[76]
accumulation of genomic mutations that may culminate in tumour growth . Mutations in TP53 are
associated with poor prognosis and are more common in cases with unmutated IGHV genes [29,31,36,38,56] .

Other low prevalence genes cluster into key biological pathways
Additional recurrently mutated genes are continuing to emerge from high-throughput sequencing studies,
though at lower frequencies [Table 2 and Figure 3]. As previously noted, these genes often cluster within
biological pathways of known importance to B-cell differentiation and in cancer more generally. Some of
the most well-established genes and pathways are summarized below:

NF-κB signalling plays an essential role in MZ B-cell development and differentiation . When normal B
[77]
lymphocytes respond to antigens, NF-κB signalling is activated, reprogramming cells to favour cell cycle
progression, survival, cytokine secretion and inflammation [78,79] . NF-κB activation, through either the
canonical or non-canonical pathway, is transient in normal cells and depends on external stimuli including
[78]
ligands for the BCR and for the Toll-like receptors , while termination of signalling is dependent on
negative feedback mechanisms including re‐accumulation of IκBα and induction of A20 . Both unbiased
[79]
and targeted approaches have uncovered molecular lesions affecting genes belonging to the NF-κB pathway,
as well as upstream pathways connected to NF-κB activation . Most notably TNFAIP3 (A20), a negative
[78]
regulator of NF-κB signalling, has been found mutated in ~13% of SMZL cases [23,24,29,32,56,57,80] . Parry et al.
[24]
also reported higher frequency of TNFAIP3 mutations in cases that subsequently transformed, along with
truly unmutated IGHV genes. Other negative regulators harbouring mutations include TRAF3 (8%) and
BIRC3 (5%-11%). Both TRAF3 and BIRC3 are part of the regulatory system that negatively regulates
MAP3K14, a central activator of noncanonical signalling and another target of mutations in SMZL .
[32]
Activating mutations in positive regulators have also been found mainly in CARD11 (~5%) and IKBKB
(~3%). Furthermore, mutations in this pathway seem to be mutually exclusive pointing to multiple
independent molecular mechanisms targeting NF-κB signalling in SMZLs .
[32]