Recent advances in understanding the biology of marginal zone lymphoma

There are three different marginal zone lymphomas (MZLs): the extranodal MZL of mucosa-associated lymphoid tissue (MALT) type (MALT lymphoma), the splenic MZL, and the nodal MZL. The three MZLs share common lesions and deregulated pathways but also present specific alterations that can be used for their differential diagnosis. Although trisomies of chromosomes 3 and 18, deletions at 6q23, deregulation of nuclear factor kappa B, and chromatin remodeling genes are frequent events in all of them, the three MZLs differ in the presence of recurrent translocations, mutations affecting the NOTCH pathway, and the transcription factor Kruppel like factor 2 ( KLF2) or the receptor-type protein tyrosine phosphatase delta ( PTPRD). Since a better understanding of the molecular events underlying each subtype may have practical relevance, this review summarizes the most recent and main advances in our understanding of the genetics and biology of MZLs.


Introduction
In the World Health Organization classification, there are three different marginal zone lymphoma (MZL) entities with specific diagnostic criteria, behavior, and therapeutic implications: the extranodal MZL of mucosa-associated lymphoid tissue (MALT) type (MALT lymphoma), the splenic MZL (SMZL), and the nodal MZL (NMZL) 1 . MALT lymphoma is the commonest MZL type, accounting for 5 to 8% of all B-cell lymphomas 2,3 . Their differential diagnosis is not straightforward in the non-rare cases presenting with disseminated disease involving lymph nodes, spleen, peripheral blood, bone marrow, or other extranodal sites. A better understanding of the molecular events underlying each subtype may have practical relevance.
MZLs are believed to derive from B cells of the "marginal zone", the external part of the secondary lymphoid follicles. The marginal zone is more evident in the lymphatic tissues continuously exposed to external antigens, such as the mesenteric lymph nodes, the MALT, and the spleen. Marginal zone B cells act as innate-like lymphocytes able to mount rapid antibody responses to both T cell-dependent and T cell-independent antigens, mostly the latter 4 .
We will now highlight the most recent and main advances in our understanding of the genetics and biology of MZLs.

NF-κB signaling
Active NF-κB signaling is necessary for the generation and maintenance of normal marginal zone B cells and this requires weak B-cell receptor (BCR) signaling (for example, started by auto-antigens and leading to canonical NF-κB pathway activation) or CD40 signaling, activating the non-canonical NF-κB pathway 36,37 . Following BCR engagement, Src family kinases phosphorylate the cytoplasmic ITAM portions of CD79A and CD79B [38][39][40][41][42][43][44][45] . The latter bind the tyrosine kinase SYK and start a signaling cascade that, via the Bruton's tyrosine kinase (BTK), results in phosphorylation and activation of CARD11. CARD11, BCL10, and MALT1 form the CBM signaling complex linking BCR signaling to the canonical NF-κB pathway. Upon phosphorylation, CARD11 acquires an open conformation, allowing the recruitment of CARD11 to MALT1 and BCL10 into the CBM complex and activate the IKBKB kinase. IKBKB phosphorylates the IκBα inhibitor molecule, causing its proteasome-mediated degradation. Finally, the NF-κB complexes (mainly p50/RelA and p50/c-Rel dimers) can enter the nucleus and act as transcriptional factors. TNFAIP3 negatively regulates the whole pathway, adding and subtracting ubiquitin moieties to different NF-κB signaling pathways. Binding of CD40 activates the non-canonical NF-κB pathway. Following disruption of a negative regulatory complex comprising TRAF3/MAP3K14-TRAF2/BIRC3, the MAP3K14 kinase (also known as NIK) phosphorylates NFKB2 (p100), causing its proteasomal processing and the formation of p52-containing NF-κB dimers. In particular, BIRC3 (cIAP2), owing to its C-terminal RING domain, has ubiquitin ligase (E3) activity 46 and leads to BCL10 and MAP3K14 ubiquitination 46 . Similarly, TRAF3 induces MAP3K14 degradation by recruiting it to the BIRC3 ubiquitin ligase complex. The p52 protein dimerizes with RelB to translocate into the nucleus, acting as a transcriptional factor.
The t(14;18) translocation occurs in 15 to 20% of MALT lymphomas, more frequently in non-gastrointestinal sites such as lung and ocular adnexa, and brings the intact MALT1 gene under the control of the IGH enhancer, resulting in deregulated expression of MALT1 directly contributing to NF-κB activation 10,54 . The t(1;14) translocation and its variant t(1;2)(p22;p12) occur in 1 to 2% of MALT lymphomas 55 . Similarly to the t(14;18), the entire coding region of BCL10 is moved under the control of the IGH enhancer region (or the IGLk region in the case of a variant translocation) and has a direct effect on the NF-κB signaling 56 .
The important role of BIRC3-MALT1 fusion protein, as well as MALT1 and BCL10 upregulation, in MALT lymphoma is further underlined by mouse models with development of MALT lymphomas and DLBCL in MALT1 gene transgenic mice 57 , expansion of marginal zone cells in BIRC3-MALT1 18,35, and BCL10 transgenic mice 58 .
In SMZL and NMZL, NF-κB signaling is also sustained by mutations occurring in genes coding members of upstream pathways, such as Toll-like receptor (TLR) and BCR signaling.

NOTCH signaling
Similarly to NF-κB signaling, NOTCH activation is important for marginal zone differentiation and homing of B cells to the splenic marginal zone 72-74 . The NOTCH2 gene is mutated in 10 to 25% of SMZLs, in about 25% of NMZLs, and in less than 5% of MALT lymphomas 6,32,33,52,68,75,76 . NOTCH1 is also mutated in about 5% of SMZLs but not at all or at a much lower frequency in NMZL and MALT lymphomas 6,77 . Negative regulators of NOTCH signaling (such as SPEN, DTX1, and MAML2) are also mutated, though at lower frequency, bringing NOTCH activation by genetic events to 40% of SMZLs and NMZLs 6,32,52 . NOTCH2 and NOTCH1 are heterodimeric transmembrane proteins that, after binding with their ligands, undergo a cleavage of their intracellular portions, which, once in the nucleus, regulate gene expression via binding with transcriptional co-factors 78,79 . Importantly, NOTCH1 and NOTCH2 mutations cluster in the C-terminal PEST domain and cause a protein truncation with loss of the region necessary for inactivation via proteasomal degradation. Thus, mutations are believed to enhance the stability of the active NOTCH intracellular domains (NICDs) once it has been triggered by microenvironmental interactions 80 .

KLF2
Inactivating mutations in the KLF2 gene are very frequent in SMZL (20-40%) and NMZL (20% of cases) 52,81,82 . KLF2 is a transcription factor, and mice with a B cell-specific deletion of KLF2 have an increased number of splenic marginal zone B cells 83 . In lymphoma cells, mutated KLF2 delocalizes from the nucleus into the cytoplasm and is not able to inhibit the NF-κB signaling activated by upstream pathways, including the BCR and TLR pathways 82 .

PTPRD
PTPRD is a receptor-type protein tyrosine phosphatase expressed in normal germinal center B cells and, at lower levels, in marginal zone B cells 52 . Almost exclusively in NMZL, PTPRD is inactivated by mutations or deletions in about 20% of cases 52 . PTPRD regulates many biologic pathways, and NMZL cases with mutated PTPRD appear to have an increased cell proliferation, indicating an involvement of PTPRD in cell proliferation 52 .

Chromatin remodeling and epigenome regulation
As a whole, mutations in genes coding for epigenetic regulators are found in about 40% of MZLs. Although their precise consequences in MZL cells are still unknown, mutations in genes such as KMT2D (MLL2), SIN3A, ARID1A, EP300, CREBBP, and TBL1XR1 highlight a deregulation of the epigenome in all three MZLs 6,26,33,52,68,76,84 . The importance of epigenetic changes is also underlined by methylation changes described in SMZL, which associate with silencing of different tumor suppressor genes and over-expression of genes involved in BCR/PI3K/AKT/ NF-κB signaling, PRC2-complex (EZH2, EED, and SUZ12), and MYC and IRF4 targets. Clinically, epigenetic changes in SMZL associate with inferior outcome and risk of transformation to a diffuse large B-cell lymphoma (DLBCL) 85 . In MALT lymphomas, promoter methylation seems to increase with a continuum from MALT lymphoma, to MALT lymphoma with large cell component, to DLBCL. Consistently, a series of tumor suppressor genes such as CDKN2A, DAPK1, CDH1, and TNFAIP3 are silenced via promoter methylation in MALT lymphoma progression 86-88 .

Antigen stimulation
There is a lot of evidence supporting the notion that antigen stimulation is important for the development and progression of MZLs. MALT lymphoma arises from B cells within populations of immune cells induced by a chronic inflammation taking place in extranodal sites in organs that are physiologically devoid of germinal centers. The most frequent site of MALT lymphoma is the stomach, where the disease has been very clearly associated with the chronic gastritis induced by Helicobacter pylori 89 . MALT lymphomas arising in other anatomical sites have also been associated with additional infectious agents, although the etiologic link is not as strong as for the gastric localization and H. pylori 89 . These include Clamydophila psittaci in orbital adnexa MALT lymphoma 90-97 , Borrelia burgdorferi in cutaneous MALT lymphoma 98-100 , Campylobacter jejuni in immunoproliferative small intestine disease 101,102 , Achromobacter xylosoxidans in pulmonary MALT lymphoma 103 , and hepatitis C virus (HCV) in all MZLs 104-108 . Besides infection, chronic inflammations in the context of autoimmune disorders, such as Sjögren syndrome or Hashimoto's thyroiditis, are strongly associated with the development of MALT lymphomas affecting salivary glands and thyroid, respectively [109][110][111][112][113][114][115][116][117] . Besides the continuous antigenic stimulation, oncogenic events, such as those presented above, contribute to lymphoma growth and progression up to the development of frank tumor independent of the antigenic drive 89 .
MZLs present somatically mutated immunoglobulin heavy variable (IGHV) genes in nearly all cases with a pattern of somatic hypermutation and rearrangements indicative of an antigen selection [118][119][120][121][122][123][124][125] . The presence of the so-called ongoing mutations (intraclonal variation) and the biased usage of some IGHV segments indicate that the expansion of lymphoma cells could still be antigen-driven. In MALT lymphomas, there is an apparently biased usage of different IGHV families in cases derived from different anatomical sites or with particular clinical and genetic features: IGHVH1-69 in salivary gland lymphomas, IGHVH3-30 or IGHVH3-23 in gastric MALT lymphomas responsive to H. pylori eradication and without the t(11;18) translocation, IGHVH4-34 in orbital adnexal lymphomas, IGHV3 and IGHV4 families in pulmonary lymphomas, and IGHVH1-69 or IGHVH4-59 in cutaneous lymphomas 126 . Similarly, a biased IGHV usage is present in SMZL with a stereotyped BCR in about 10% of cases 127 and a biased usage of the IGHV 1-2*04 allele in about 30% of cases 123,127-130 and in NMZL with a biased usage of the IGHV4-34 gene in 20 to 30% of cases 131,132 . IGHVH1-69 is also frequently detected in HCV-related MZLs, similarly to what observed in other HCV-related B-cell expansions such as in the monoclonal rheumatoid factor-like IgM component of the type II mixed cryoglobulinemia, and in monoclonal paraproteins from patients with HCV infection [106][107][108]133 . Finally, the antibodies expressed by MALT lymphoma and SMZL cells often recognize self-antigens [134][135][136] .

Clinical implications
Molecular lesions may be of help to inform MZL diagnosis, prognosis, and therapeutic targeting. In general, the presence of trisomies of 3 and 18 as single lesions or associated only with TNFAIP3 loss or 7q deletions is highly indicative of MZL more than other small cell lymphomas. The presence of translocations affecting MALT1 and BIRC3 is basically exclusive to MALT lymphoma, in which they are associated with lower response rate to antibiotics treatment. From a diagnostic standpoint, NOTCH2 mutations are highly specific for SMZL and NMZL among mature B-cell tumors, including conditions that look alike, thus representing a biomarker with positive predictive value for non-MALT MZL specification. Within non-MALT MZL, PTPRD mutations are enriched in NMZL and thus may represent a genetic biomarker that, though not highly sensitive, is provided with a positive predictive value for NMZL specification.
From a prognostic standpoint, KLF2 mutations and NOTCH2 mutations represent promising prognostic biomarkers associated with poor survival and transformation to aggressive lymphoma whose broad application in clinical practice requires the assessment of whether their incorporation into the currently available clinical prognostic models improves risk stratification of patients.
Molecular aspects of MZL point to deregulated cellular programs worth exploring as therapeutic targets. Pharmacologic interference of NOTCH signaling, non-canonical NF-κB signaling, or upstream pathways that are connected to NF-κB, including BCR signaling, are attractive approaches in these lymphomas.

Competing interests
The authors declare that they have no competing interests.

Grant information
The author(s) declared that no grants were involved in supporting this work.

orbital and ocular adnexal lymphomas identifies frequent alterations in MYD88
Open Peer Review Current Referee Status: Editorial Note on the Review Process are commissioned from members of the prestigious and are edited as a F1000 Faculty Reviews F1000 Faculty service to readers. In order to make these reviews as comprehensive and accessible as possible, the referees provide input before publication and only the final, revised version is published. The referees who approved the final version are listed with their names and affiliations but without their reports on earlier versions (any comments will already have been addressed in the published version).
The referees who approved this article are:

Version 1
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias You can publish traditional articles, null/negative results, case reports, data notes and more The peer review process is transparent and collaborative