Best Practice & Research Clinical Haematology 30 (2017) 24e31

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Best Practice & Research Clinical Haematology

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Methylation patterns in marginal zone lymphoma

Alberto J. Arribas a, *, Francesco Bertoni a, b
a
Lymphoma & Genomics Research Program, Institute of Oncology Research (IOR), Bellinzona, Switzerland
b
Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland

a b s t r a c t

Keywords: Promoter DNA methylation is a major regulator of gene expression and transcription. The
Lymphoma identification of methylation changes is important for understanding disease pathogenesis,
Methylation for identifying prognostic markers and can drive novel therapeutic approaches. In this
Decitabine
review we summarize the current knowledge regarding DNA methylation in MALT lym-
Marginal zone
phoma, splenic marginal zone lymphoma, nodal marginal zone lymphoma. Despite
MALT
CIMP
important differences in the study design for different publications and the existence of a
sole large and genome-wide methylation study for splenic marginal zone lymphoma, it is
clear that DNA methylation plays an important role in marginal zone lymphomas, in which
it contributes to the inactivation of tumor suppressors but also to the expression of genes
sustaining tumor cell survival and proliferation. Existing preclinical data provide the
rationale to target the methylation machinery in these disorders.
© 2016 Elsevier Ltd. All rights reserved.

Promoter DNA methylation is a well-studied major regulatory machinery of gene expression and transcription [1e4].
High-throughput technologies [5e8] have provided a new insight on DNA methylation indicating a higher degree of plasticity
than previously considered [9e11]. Cell differentiation and commitment require dynamic changes in the expression of crucial
transcription factors, which are frequently orchestrated by epigenetic reprogramming [12,13]. The latter largely acts on areas
surrounding promoters and enhancers in the framework of CpG islands [14]. In hematopoietic homeostasis, methylation
patterns regulate development and maturation driving multipotent progenitors to the more mature stages of B-cells [15].
Aberrant DNA methylation not only contributes to the pathogenesis of many cancers but can also provide prognostic and/or
diagnostic markers [4,16e18]. Interestingly, among different types of cancers, there are groups of tumors that are charac-
terized by hypermethylation of multiple CpG islands, a phenomenon called “CpG Island Methylator Phenotype” (CIMP) [19].
Depending on the specific tumor type, the CIMP phenotype can be associated to an inferior or superior clinical outcome and
be strictly linked or not to the presence of precise genetic features [19]. In addition, hematological neoplasms harbor highly
recurrent somatic mutations and genomic aberrations affecting genes coding epigenetic regulators and histone modifying
enzymes suggesting a potential oncogenic role of these alterations [4,20]. Since pharmacological intervention may revert the
aberrant methylation program, novel therapeutic approaches can be explored. Furthermore, the outcome of patients affected
with B-cell lymphoma can be affected by DNA methylation, so identification of subsets of cases harboring distinct methylation
patterns might be relevant in optimizing therapy. In this review we will summarize the current knowledge regarding DNA
methylation in marginal zone lymphoma (MZLs). MZLs comprise three different entities: extranodal marginal zone lym-
phoma of mucosa-associated lymphoid tissue (MALT lymphoma), splenic marginal zone lymphoma (SMZL), nodal marginal

* Corresponding author. IOR Institute of Oncology Research, via Vincenzo Vela 6, 6500 Bellinzona, Switzerland. Fax: þ41 91 8200 397.
E-mail addresses: alberto.arribas@ior.iosi.ch (A.J. Arribas), frbertoni@mac.com (F. Bertoni).

http://dx.doi.org/10.1016/j.beha.2016.09.003
1521-6926/© 2016 Elsevier Ltd. All rights reserved.
 A.J. Arribas, F. Bertoni / Best Practice & Research Clinical Haematology 30 (2017) 24e31 25

zone lymphoma (NMZL), which share clinical and genetic features but present peculiarities that make them three distinct
disorders [21e25].

Methylation patterns in normal B-cells

The gene expression profile of MZL resembles the transcriptional programs of non-tumoral B-cells residing in the marginal
zone and of memory B-cells [26,27] (both have been postulated as the normal counterpart). The methylation patterns across
the B-cell maturation lineage from hematopoietic progenitor to long-lived plasma cells have been characterized in studies
applying Whole Genome Bisulfite Sequencing (WGBS) and high-density microarrays [13,28]. DNA methylation reprogram-
ming is necessary for all the maturation steps of B-cells, which present specific methylation profiles for each differentiation
stage [13,28]. B-cells show an overall decrease in their methylation load along their maturation lineage [13,28], and the most
pronounced methylation changes occur when germinal center (GC) B-cells differentiate to memory B-cells and/or to plasma
B-cells, when hypomethylation reprogramming affects promoters of genes crucial for B-cell development, such as ARID3A,
BCL2, BLK, EBF1 and IRF4 [28]. Dynamic changes in methylation regulate both the expression of transcription factors and of
their target genes. There is an inverse correlation between enhancer demethylation and expression of important B-cell
transcription factors such as BCL11A, EBF1, IRF4, MEF2, PAX5 and TCF3 [28], but there are also methylation changes at the
binding sites of different transcription factors, including NF-kB, OCT2, IRF4, AP-1, EBF1 and RUNX3 transcription factors [13].
Conversely, dynamic DNA methylation does not seem to play a major role in controlling the expression of genes involved in B-
cell receptor signaling genes [28]. Although memory B-cells show strong methylome reprogramming when compared to GC
B-cells, the observed changes would affect mostly Polycomb-repressed areas (harboring tri-methylation at lysine 27 of
histone 3, H3k27me3) both in CpG and non-CpG regions [28], and the hypermethylation of Polycomb-repressed areas could
be due to de novo methylation since DNMT3A is up-regulated in memory B-cells while DNMT1 (maintenance methylation)
shows lower levels of expression when compared to GC B-cells [28].

Methylation: from normal to tumoral cells

Epigenetic reprogramming is a hallmark of cancer, including lymphomas [1e4,16,29e44], determining in many cases
high-methylation of Polycomb-repressed areas and/or low-methylation of heterochromatin [1,4,45e47]. Neoplastic B-cells
harbor a methylation pattern similar to the one observed in non-tumoral memory B- and plasma cells [28]. Martin-Subero and
coworkers reported a microarray-based comprehensive study on DNA methylation of a large cohort of the most represen-
tative tumors derived from B-cells (n ¼ 203, including 10 MALT lymphoma specimens), T-cells (n ¼ 30), and myeloid cells
(n ¼ 134), as well as non-tumoral controls from different hematopoietic cell types (n ¼ 37) [48]. In normal B-cells, low-
methylation affects not only B-cell specific genes, such as BLK, SYK and CD82, but also transcription factors and pro-
survival genes (RUNX3, LCK, MET, CCL3, DDR1 and PI3K genes) [48]. Over 200 genes are hypermethylated in at least one
condition and, interestingly, six genes (DBC1, DIO3, FZD9, HS3ST2, MOS and MYOD1) are significantly hypermethylated in all
the hematological cancers. Interestingly, MALT lymphomas exhibit low levels of de novo DNA methylation, with only three
genes (BMP4, LMO2 and SFTPB) being significantly hypermethylated, and none hypomethylated. Neoplasms believed to derive
from GC B-cells [diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma and follicular lymphoma] are characterized by a
higher number of hypermethylated genes than other lymphoid malignancies like mantle cell lymphoma (MCL), B-cell chronic
lymphocytic leukemia (CLL) and MALT lymphoma suggesting that lymphoid tumors arising from GC cells tend to acquire
higher levels of de novo DNA methylation [48]. Mechanisms underlying aberrant methylation in cancer are not well clarified
yet [49]. Nevertheless, somatic mutations and genomic lesions affecting epigenetic machinery are a frequent event in he-
matological malignancies including MZL [20,50e54]. The most common genomic lesions (somatic mutation and copy
number variations together) affecting chromatin remodeling genes identified so far in MZL are summarized in Table 1.
Nonetheless, the most recurrent genomic lesions in MZL, those affecting KMT2D (MLL2) and CREBBP genes, and ARID1A and

Table 1
Most commonly affected chromatin remodeling genes in MZL.

Gene SMZL [50] SMZL [51] SMZL [53] SMZL [52] NMZL [54]

n ¼ 40 n¼7 n ¼ 14 n ¼ 175 n ¼ 35
MLL2 15% (6/40) 14% (1/7) e 11% (20/175) 34% (12/35)
CREBBP e 29% (2/7) 7% (1/14) 5% (9/175) 9% (3/35)
ARID family 5% (2/40) 7% (1/14) 6% (10/175) 23% (8/35)
HISTONE1 family e e 29% (4/14) e 14% (5/35)
ARID1A 5% (2/40) e e 6% (10/175) 14% (5/35)
TBL1RXR1 10% (4/40) e e e 14% (5/35)
EP300 5% (2/40) e e e 9% (3/35)
TRRAP 3% (1/40) 14% (1/7) e e 9% (3/35)
ARID1B e e e e 9% (3/35)
SMARC family e e 7% (1/14) e 3% (1/35)
SIN3A 8% (3/40) e e e e
26 A.J. Arribas, F. Bertoni / Best Practice & Research Clinical Haematology 30 (2017) 24e31

Histone1 families, seem to be not restricted to MZL and in fact they are common events across lymphoma subtypes
comprising DLBCL, follicular lymphoma, MCL and CLL. Hence, the aberrations in epigenetic modifiers are not specific driver
lesions for MZL, but they appear as common early events in lymphomagenesis [55].

MALT lymphoma

The information we currently have on the role of promoter methylation in MALT lymphoma comes from studies typically
investigating a small number of genes and/or based on small series of cases [42e44,56e71]. Moreover, methylation data can
be heavily affected by the normal cells present in the analyzed tissue, thus it is difficult to properly compare the data obtained
for MALT lymphomas arising from different anatomical sites and from different studies, which did not always include the
most appropriate normal controls.
The first studies on DNA promoter methylation including MALT lymphomas are from the late nineties [42e44,56e62], and
investigated one or very few key tumor suppressor genes mostly using methyl-specific-PCR (MS-PCR). More recently, larger
number of genes have been studied (Table 2) [63e70], but, so far, only one study of genome-wide methylation profiling has
been dedicated only to MALT lymphoma [71].
A few tumor suppressor genes, commonly methylated also in other cancers [19,72], are methylated in MALT lymphomas: i)
CDKN2A gene, coding the cyclin dependent kinase inhibitor 2A p16INK4A (median 42% among studies, range 20e78%)
[63,64,67,69,70,73]; ii) DAPK1 gene, coding the death-associated protein kinase 1 (42% among studies, range 20e78%)
[63,64,67,69,70,73]; MGMT gene, coding the O-6-methylguanine-DNA methyltransferase (25%, 0%e78%) [63,65,67,69,70,73];
CDH1 (44%. 17%e77%) [63,69,70,73] (Table 2).
Since Helicobacter pylori -associated chronic gastric inflammation induces aberrant methylation in epithelial cells leading
to an increased risk of gastric cancer [74], the relationship between DNA promoter methylation and H. pylori infection in
gastric MALT lymphoma and Chamydophila psittaci in ocular adnexal MALT lymphoma have been investigated. Six C. psittaci
positive cases were compared to six negative cases for the promoter methylation status of approximately 14,000 genes: 184
CpG islands were differentially methylated comprising 75 hypermethylated probes and 109 hypomethylated in C. psittaci
positive subset [71]. The differentially methylated genes were mainly represented by genes involved in immunity, signal
transduction biological process and receptor, ion channel and extracellular matrix [71]. Differential methylation according to
responsiveness to doxycycline, which is not active only in C. psittaci positive cases but also in negative [75], showed 778 CpG
sites differentially methylated: 442 hypermethylated and 336 hypomethylated loci in the responders, regulating signal
transduction process and receptor, transcription factor and extracellular matrix molecular functions [71]. Among genes
potentially regulated at methylation level, DUSP22, hypermethylated, could lead to JNK pathway activation in C. psittaci
positive tumors, while IRAK1 and CXCL6, hypo- and hyper-methylated, respectively, in responders, might be directly involved
in the mechanism of action of doxycycline [71]. In another study of 35 ocular adnexal MALT lymphomas from South Korea,
methylation in CpG islands of 11 genes [DAPK1, CDH1, MT1G, THBS1, RARB, CDKN2A (p16, p14), MGMT and RASSF1A] was
evaluated [69], and promoters for DAPK1 and CDH1 were frequently methylated in tumors but not in normal samples
(no. ¼ 13) and the latter was correlated with C. psittaci infection. In the stomach, DNA promoter methylation appeared
correlated to H. pylori positivity in a series of 21 gastric MALT lymphomas [67], but not in a smaller one of 12 cases [63].
Interestingly, the frequency of methylated genes seems to increase along the progression of disease with a continuum from
the healthy donors to MALT lymphoma, to MALT lymphoma with large cell component and to DLBCL [67]. This was shown in a
study that investigated 11 genes (CDKN1C, CDKN2A, MLH1, CDKN2B, TP73, MGMT, DAPK1, MINT1, MINT2, MINT31 and HCAD) in
gastric MALT lymphomas (no. ¼ 21), MALT lymphoma with large cell component (no. ¼ 5) and DLBCL (no. ¼ 15) [67].
In gastric MALT lymphoma, the presence of t(11;18) (q21;q21) appears inversely correlated with CDKN2A hyper-
methylation in gastric MALT lymphoma, while BCL10 expression is associated with MAD2 promoter methylation in a series of
gastric MALT lymphoma [64].
The negative regulator of nuclear factor kappa B (NFkB) TNFAIP3 (A20) is a tumor suppressor commonly inactivated in a
25% of MZL by deletion or mutation and specifically affects 36% of MALT lymphoma patients [76]. Epigenetics could represent
an alternative mechanism of TNFAIP3 inactivation as reported in 7/27 MALT lymphomas of the ocular adnexa [68], although
no promoter methylation was detected in additional 10 cases from another series [76].

Splenic marginal zone lymphoma

A large study, based on a training (n ¼ 98)/validation (n ¼ 36) approach integrating methylation with gene expression, copy
number variations and clinical features, has identified two groups of SMZL cases [77]. One group comprises approximately 20%
of all the cases and exhibits a high level of general DNA promoter methylation, compatible with a CIMP-positive phenotype,
significantly inferior overall survival than the other one (Low-M), higher frequency of IGHV1-02 usage, of NOTCH2 gene somatic
mutations, of 7q31-32 loss and of histological transformation to DLBCL [77]. Importantly, the methylation status of only three
genes (KLF4, CACNB2 and HTRA1) is able to highly predict the CIMP-positive phenotype and the outcome [77]. CIMP-positive
cases are also characterized by a specific gene expression signature: i) low expression of PRC2-complex targets, genes
harboring H3K27me3 and H3K4me3 marks, chromatin remodeling genes and targets repressed by CDH1, TP53 and TP63 tumor
suppressor genes, and also of genes coding for tumor suppressors including KLF4, DAPK1, CDKN2D (p19), CDKN1C (p57), CDH1,
CDH2, SPRY2, CBX1, WT1 and TIMP3; ii) upregulation of pro-survival genes (TCL1A/B, IL2RB, BCL2L10, CARD11, IFGN, UBD and
Table 2
Frequency of DNA promoter methylation in genes studied with Methylation-Specific PCR in different series of MALT lymphoma samples.

Gastric MALT Gastric MALT Gastric MALT Gastric MALT Salivary glands Orbital adnexa Orbital adnexa Cutaneous MALT Thymic MALT
lymphoma [63] lymphoma [64] lymphoma, lymphoma, MALT lymphoma MALT lymphomaa MALT lymphoma lymphoma [73] lymphoma
H. pylori e [67] H. pylori þ [67] [65] [68] [69] [70]

n ¼ 12 n ¼ 40 n ¼ 12 n¼9 n¼8 n ¼ 27 n ¼ 35 n ¼ 60 n ¼ 18
CDKN2A 42% (5/12) 75% (30/40) 42% (5/12) 78% (7/9) e e 20% (7/35) 49% (26/53) 28% (5/18)
(p16, INK4A)
DAPK1 17% (2/12) e 58% (7/12) 100% (9/9) 63% (5/8) e 94% (33/35) 43% (25/58) 61% (11/18)
MGMT 58% (7/12) e 25% (3/12) 78% (7/9) 0% (0/8) e 6% (2/35) 15% (9/60) 39% (7/18)
CDH1 (ECAD) 17% (2/12) e e e e e 77% (27/35) 39% (14/36) 50% (9/18)
MLH1 (hMLH1) 50% (6/12) e 0% (0/12) 0% (0/9) e e e e e
ATM 17% (2/12) e e e e e e e e
CDKN1C e e 42% (5/12) 56% (5/6) e e e e e
(p57, KIP2)
CDKN2B e e 8% (1/12) 11% (1/9) e e e e e
(p15, INK4B)
TP73 e e 17% (2/12) 11% (1/9) 25% (2/8) e e e e
MINT1 e e 17% (2/12) 11% (1/9) e e e e e
MINT2 e e 25% (3/12) 67% (6/9) e e e e e
MINT31 e e 25% (3/12) 56% (5/6) e e e e e
CDH13 (HCAD) e e 83% (10/12) 89% (8/9) e e e e e
RARB e e e e e e 31% (11/35) 21% (7/34) 28% (5/18)
p14 ARF, CDKN2A e e e e e e 0% (0/35) 48% (29/60) 50% (9/18)
TIMP3 e e e e e e e 9% (5/58) 33% (6/18)
MAD2 e 61% (23/38) e e e e e e e
TNFAIP3 (A20) e e e e e 26% (7/27) e e e
MT1G e e e e e e 49% (17/35) e e
THBS1 e e e e e e 37% (13/35) e e
A.J. Arribas, F. Bertoni / Best Practice & Research Clinical Haematology 30 (2017) 24e31

RASSF1A e e e e e e 0% (0/35) e e
a
Including two cases of salivary glands MALT lymphoma.
27
28 A.J. Arribas, F. Bertoni / Best Practice & Research Clinical Haematology 30 (2017) 24e31

PIK3CB), transcripts coding for PRC2-complex (EZH2, EED and SUZ12), MYC and IRF4 targets and genes involved in B-cell re-
ceptor/PI3K/AKT/NFkB signaling [77]. Parallel work performed on in vitro SMZL models treated with the demethylating agent
decitabine shows the latter is, at least partially, able to reverse the CIMP-positive phenotype, re-expressing tumor suppressors
(such as KLF4, SOCS1, NFKBIA, CDKN1A, CDKN2D, KAT2B, CYLD, DAPK1 and FBXW4) and downregulating genes sustaining cell
survival and proliferation (MYC, BCL2, STAT3/5 and CD40) [77]. Thus, the combination of analyses on clinical specimens with
in vitro experiments has allowed the identification of the important role of methylation in SMZL and has proposed the use of a
demethylating agent in this disorder as a single agent [77,78], a possibility further strengthened by recent in vivo data, or in
combination with compounds targeting the other deregulated pathways (Notch, BCR signaling, EZH2) [79].
Another group has explored the possible contribution of methylation to the recurrent 7q deletion, studying, in 12 cases,
the methylation stat us of 18 transcripts that are down-regulated in association with the presence of the genomic aberration
[80]. The down-regulation could be due to methylation in 10 transcripts (CPA4, OPN1SW, NAG8, LRRC4, CPA5, CPA2, TSGA13,
CPA1, C7orf45 and NYD-SP18) but this phenomenon does not seem to be specific for 7q- SMZL cases, being detected also in
other lymphoid tumors [80].

Nodal marginal zone lymphoma

No study has specifically addressed the role of DNA methylation in NMZL. However, promoter methylation contributes to
the downregulation of PTPRD, whose role as a tumor suppressor in NMZL has been discovered by applying next-generation
sequencing in a series of 35 patients [54].

Conclusions and take home messages

In this review we summarized the most relevant findings on DNA promoter methylation reported so far in MALT lym-
phoma, SMZL and NMZL and their postulated normal counterparts, although important differences in study design (assays
used to detect methylation, samples size, background of normal cells) do not allow a proper comparison of the results derived
from the different studies. Methylation plays an important role in the control of normal B cells [13,28]. In contrast to their
normal counterparts, non-GC B-cell lymphomas, including MZLs, exhibit low levels of de novo DNA methylation when
compared to GC-derived neoplasms [48], MZLs subtypes bear promoter methylation changes that lead to the inactivation of a
series of tumor suppressors but also to the activation of genes that sustain the lymphoma cell survival and proliferation. In
SMZL, a CIMP-phenotype is associated with inferior outcome. Importantly, available evidence indicates that the lymphoma-
associated aberrant methylation might be pharmacologically targeted.

Summary

DNA promoter methylation is fundamental in controlling gene expression and transcription in normal and neoplastic cells.
Lymphomas frequently bear genetic lesions that affect genes encoding epigenetic regulators and histone modifying enzymes,
underlying the importance of the epigenome in these diseases. The study of DNA methylation in tumor cells helps in the
understanding of the disease, in providing prognostic markers and therapeutic approaches. The literature on the role of
methylation in marginal zone lymphomas is patchy with only a few papers addressing the topic in a genome-wide way, and
larger studies are still needed. However, available literature shows that DNA methylation clearly plays a role in marginal zone
lymphomas, contributing to the inactivation of tumor suppressors but also to the expression of cancer genes. Also, in SMZL, a
CIMP-phenotype is associated with inferior outcome. Further preclinical and clinical efforts are indicated to explore the
possible therapeutic implications of aberrant methylation in marginal zone lymphomas.

Conflict of interest

The Authors have no conflict of interest regarding data presented in this manuscript.

Practice Points

 DNA promoter methylation is fundamental to control gene expression and transcription in normal and neoplastic
cells.
 The study of DNA methylation helps in the understanding of the disease, in providing prognostic markers and
therapeutic approaches.
 DNA methylation clearly plays a role in marginal zone lymphomas, contributing to the inactivation of tumor
suppressors but also to the expression of cancer genes.
 In SMZL, a CIMP-phenotype is associated with inferior outcome, and preclinical evidences suggest demethylating
agents as potential therapeutic modalities.
 A.J. Arribas, F. Bertoni / Best Practice & Research Clinical Haematology 30 (2017) 24e31 29

Research Agenda

 The literature on the role of methylation in marginal zone lymphomas is patchy, and larger and dedicated studies
are still needed.
 Further preclinical and clinical efforts are indicated to explore the possible therapeutic implications of aberrant
methylation in marginal zone lymphomas.

Acknowledgements

This work was partially based on results obtained thanks to a research grant from the Fondazione Ticinese per la Ricerca
sul Cancro. The authors thank their colleague Afua Adjeiwaa Mensah for editing the article.

References

[1] Allis CD, Jenuwein T. The molecular hallmarks of epigenetic control. Nat Rev Genet 2016;17(8):487e500.
[2] Paluch BE, Naqash AR, Brumberger Z, Nemeth MJ, Griffiths EA. Epigenetics: a primer for clinicians. Blood Rev 2016;30(4):285e95.
[3] Yen CY, Huang HW, Shu CW, Hou MF, Yuan SS, Wang HR, et al. DNA methylation, histone acetylation and methylation of epigenetic modifications as a
therapeutic approach for cancers. Cancer Lett 2016;373(2):185e92.
[4] Jiang Y, Hatzi K, Shaknovich R. Mechanisms of epigenetic deregulation in lymphoid neoplasms. Blood 2013;121(21):4271e9.
[5] Sun Z, Cunningham J, Slager S, Kocher JP. Base resolution methylome profiling: considerations in platform selection, data preprocessing and analysis.
Epigenomics 2015;7(5):813e28.
[6] Wang T. The modern cancer methylomics: genome-wide DNA methylation analysis. Am Assoc Cancer Res Educ Book 2012:213e7.
[7] Soto J, Rodriguez-Antolin C, Vallespin E, de Castro Carpeno J, Ibanez de Caceres I. The impact of next-generation sequencing on the DNA methylation-
based translational cancer research. Transl Res 2016;169. 1e18.e1.
[8] Kurdyukov S, Bullock M. DNA methylation analysis: choosing the right method. Biology 2016;5(1).
[9] Ziller MJ, Gu H, Muller F, Donaghey J, Tsai LT, Kohlbacher O, et al. Charting a dynamic DNA methylation landscape of the human genome. Nature 2013;
500(7463):477e81.
[10] Suelves M, Carrio E, Nunez-Alvarez Y, Peinado MA. DNA methylation dynamics in cellular commitment and differentiation. Brief Funct Genomics 2016
Jun 8. pii: elw017.
[11] Schultz MD, He Y, Whitaker JW, Hariharan M, Mukamel EA, Leung D, et al. Human body epigenome maps reveal noncanonical DNA methylation
variation. Nature 2015;523(7559):212e6.
[12] Bock C, Beerman I, Lien WH, Smith ZD, Gu H, Boyle P, et al. DNA methylation dynamics during in vivo differentiation of blood and skin stem cells. Mol
Cell 2012;47(4):633e47.
[13] Oakes CC, Seifert M, Assenov Y, Gu L, Przekopowitz M, Ruppert AS, et al. DNA methylation dynamics during B cell maturation underlie a continuum of
disease phenotypes in chronic lymphocytic leukemia. Nat Genet 2016;48(3):253e64.
[14] Guillamot M, Cimmino L, Aifantis I. The impact of DNA methylation in hematopoietic malignancies. Trends Cancer 2016;2(2):70e83.
[15] Cedar H, Bergman Y. Epigenetics of haematopoietic cell development. Nat Rev Immunol 2011;11(7):478e88.
[16] Chambwe N, Kormaksson M, Geng H, De S, Michor F, Johnson NA, et al. Variability in DNA methylation defines novel epigenetic subgroups of DLBCL
associated with different clinical outcomes. Blood 2014;123(11):1699e708.
[17] Krajnovic M, Jovanovic MP, Mihaljevic B, Andelic B, Tarabar O, Knezevic-Usaj S, et al. Hypermethylation of p15 gene in diffuse e large B-cell lymphoma:
association with less aggressiveness of the disease. Clin Transl Sci 2014;7(5):384e90.
[18] Giachelia M, Bozzoli V, D'Alo F, Tisi MC, Massini G, Maiolo E, et al. Quantification of DAPK1 promoter methylation in bone marrow and peripheral blood
as a follicular lymphoma biomarker. J Mol Diagn 2014;16(4):467e76.
[19] Suzuki H, Yamamoto E, Maruyama R, Niinuma T, Kai M. Biological significance of the CpG island methylator phenotype. Biochem Biophys Res Commun
2014;455(1e2):35e42.
[20] Butler JS, Dent SY. The role of chromatin modifiers in normal and malignant hematopoiesis. Blood 2013;121(16):3076e84.
[21] Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri A, Stein H, et al., editors. WHO classification of tumours of haematopoietic and lymphoid tissues.
Lyon, France: IARC Press; 2008.
[22] Swerdlow SH, Campo E, Pileri SA, Harris NL, Stein H, Siebert R, et al. The 2016 revision of the World Health Organization classification of lymphoid
neoplasms. Blood 2016;127(20):2375e90.
[23] Arcaini L, Rossi D, Paulli M. Splenic marginal zone lymphoma: from genetics to management. Blood 2016;127(17):2072e81.
[24] Thieblemont C, Molina T, Davi F. Optimizing therapy for nodal marginal zone lymphoma. Blood 2016;127(17):2064e71.
[25] Zucca E, Bertoni F. The spectrum of MALT lymphoma at different sites: biological and therapeutic relevance. Blood 2016;127(17):2082e92.
[26] Arribas AJ, Campos-Martin Y, Gomez-Abad C, Algara P, Sanchez-Beato M, Rodriguez-Pinilla MS, et al. Nodal marginal zone lymphoma: gene expression
and miRNA profiling identify diagnostic markers and potential therapeutic targets. Blood 2012;119(3):e9e21.
[27] Arribas AJ, Gomez-Abad C, Sanchez-Beato M, Martinez N, Dilisio L, Casado F, et al. Splenic marginal zone lymphoma: comprehensive analysis of gene
expression and miRNA profiling. Mod Pathol 2013;26(7):889e901.
[28] Kulis M, Merkel A, Heath S, Queiro s AC, Schuyler RP, Castellano G, et al. Whole-genome fingerprint of the DNA methylome during human B cell
differentiation. Nat Genet 2015;47(7):746e56.
[29] De S, Shaknovich R, Riester M, Elemento O, Geng H, Kormaksson M, et al. Aberration in DNA methylation in B-cell lymphomas has a complex origin
and increases with disease severity. PLoS Genet 2013;9(1):e1003137.
[30] Enjuanes A, Albero R, Clot G, Navarro A, Bea S, Pinyol M, et al. Genome-wide methylation analyses identify a subset of mantle cell lymphoma with a
high number of methylated CpGs and aggressive clinicopathological features. Int J Cancer 2013;133(12):2852e63.
[31] Asmar F, Punj V, Christensen J, Pedersen MT, Pedersen A, Nielsen AB, et al. Genome-wide profiling identifies a DNA methylation signature that as-
sociates with TET2 mutations in diffuse large B-cell lymphoma. Haematologica 2013;98(12):1912e20.
[32] Clozel T, Yang S, Elstrom RL, Tam W, Martin P, Kormaksson M, et al. Mechanism-based epigenetic chemosensitization therapy of diffuse large B-cell
lymphoma. Cancer Discov 2013;3(9):1002e19.
[33] Bethge N, Honne H, Hilden V, Troen G, Eknaes M, Liestol K, et al. Identification of highly methylated genes across various types of B-cell non-Hodgkin
lymphoma. PLoS One 2013;8(11):e79602.
30 A.J. Arribas, F. Bertoni / Best Practice & Research Clinical Haematology 30 (2017) 24e31

[34] Loeffler M, Kreuz M, Haake A, Hasenclever D, Trautmann H, Arnold C, et al. Genomic and epigenomic co-evolution in follicular lymphomas. Leukemia
2015;29(2):456e63.
[35] Queiros AC, Villamor N, Clot G, Martinez-Trillos A, Kulis M, Navarro A, et al. A B-cell epigenetic signature defines three biologic subgroups of chronic
lymphocytic leukemia with clinical impact. Leukemia 2015;29(3):598e605.
[36] Kulis M, Heath S, Bibikova M, Queiros AC, Navarro A, Clot G, et al. Epigenomic analysis detects widespread gene-body DNA hypomethylation in chronic
lymphocytic leukemia. Nat Genet 2012;44(11):1236e42.
[37] Rinaldi A, Mensah AA, Kwee I, Forconi F, Orlandi EM, Lucioni M, et al. Promoter methylation patterns in Richter syndrome affect stem-cell maintenance
and cell cycle regulation and differ from de novo diffuse large B-cell lymphoma. Br J Haematol 2013;163(2):194e204.
[38] Pan H, Jiang Y, Boi M, Tabbo F, Redmond D, Nie K, et al. Epigenomic evolution in diffuse large B-cell lymphomas. Nat Commun 2015;6:6921.
[39] Hopp L, Loffler-Wirth H, Binder H. Epigenetic heterogeneity of B-Cell lymphoma: DNA methylation, gene expression and chromatin states. Genes
2015;6(3):812e40.
[40] Kretzmer H, Bernhart SH, Wang W, Haake A, Weniger MA, Bergmann AK, et al. DNA methylome analysis in Burkitt and follicular lymphomas identifies
differentially methylated regions linked to somatic mutation and transcriptional control. Nat Genet 2015;47(11):1316e25.
[41] Kataoka K, Nagata Y, Kitanaka A, Shiraishi Y, Shimamura T, Yasunaga J, et al. Integrated molecular analysis of adult T cell leukemia/lymphoma. Nat
Genet 2015;47(11):1304e15.
[42] Martinez-Delgado B, Fernandez-Piqueras J, Garcia MJ, Arranz E, Gallego J, Rivas C, et al. Hypermethylation of a 50 CpG island of p16 is a frequent event
in non-Hodgkin's lymphoma. Leukemia 1997;11(3):425e8.
[43] Martinez-Delgado B, Robledo M, Arranz E, Osorio A, García MJ, Echezarreta G, et al. Hypermethylation of p15/ink4b/MTS2 gene is differentially
implicated among non-Hodgkin's lymphomas. Leukemia 1998;12(6):937e41.
[44] Rossi D, Capello D, Gloghini A, Franceschetti S, Paulli M, Bhatia K, et al. Aberrant promoter methylation of multiple genes throughout the clinico-
pathologic spectrum of B-cell neoplasia. Haematologica 2004;89(2):154e64.
[45] Schlesinger Y, Straussman R, Keshet I, Farkash S, Hecht M, Zimmerman J, et al. Polycomb-mediated methylation on Lys27 of histone H3 pre-marks
genes for de novo methylation in cancer. Nat Genet 2007;39(2):232e6.
[46] Martín-Subero JI, Kreuz M, Bibikova M, Bentink S, Ammerpohl O, Wickham-Garcia E, et al. New insights into the biology and origin of mature
aggressive B-cell lymphomas by combined epigenomic, genomic, and transcriptional profiling. Blood 2009;113(11):2488e97.
[47] Wouters BJ, Delwel R. Epigenetics and approaches to targeted epigenetic therapy in acute myeloid leukemia. Blood 2016;127(1):42e52.
[48] Martin-Subero JI, Ammerpohl O, Bibikova M, Wickham-Garcia E, Agirre X, Alvarez S, et al. A comprehensive microarray-based DNA methylation study
of 367 hematological neoplasms. PLoS One 2009;4(9):e6986.
[49] You JS, Jones PA. Cancer genetics and epigenetics: two sides of the same coin? Cancer Cell 2012;22(1):9e20.
[50] Rossi D, Trifonov V, Fangazio M, Bruscaggin A, Rasi S, Spina V, et al. The coding genome of splenic marginal zone lymphoma: activation of NOTCH2 and
other pathways regulating marginal zone development. J Exp Med 2012;209(9):1537e51.
[51] Parry M, Rose-Zerilli MJ, Gibson J, Ennis S, Walewska R, Forster J, et al. Whole exome sequencing identifies novel recurrently mutated genes in patients
with splenic marginal zone lymphoma. PLoS One 2013;8(12):e83244.
[52] Parry M, Rose-Zerilli MJ, Ljungstrom V, Gibson J, Wang J, Walewska R, et al. Genetics and prognostication in splenic marginal zone lymphoma:
revelations from deep sequencing. Clin Cancer Res 2015;21(18):4174e83.
[53] Martinez N, Almaraz C, Vaque JP, Varela I, Derdak S, Beltran S, et al. Whole-exome sequencing in splenic marginal zone lymphoma reveals mutations
in genes involved in marginal zone differentiation. Leukemia 2014;28(6):1334e40.
[54] Spina V, Khiabanian H, Messina M, Monti S, Cascione L, Bruscaggin A, et al. The genetics of nodal marginal zone lymphoma. Blood 2016 Sep 8;128(10):
1362e73.
[55] Green MR, Gentles AJ, Nair RV, Irish JM, Kihira S, Liu CL, et al. Hierarchy in somatic mutations arising during genomic evolution and progression of
follicular lymphoma. Blood 2013;121(9):1604e11.
[56] Martinez-Delgado B, Melendez B, Cuadros M, Jose Garcia M, Nomdedeu J, Rivas C, et al. Frequent inactivation of the p73 gene by abnormal methylation
or LOH in non-Hodgkin's lymphomas. Int J Cancer 2002;102(1):15e9.
[57] Kim YS, Kim JS, Jung HC, Lee CH, Kim CW, Song IS, et al. Regression of low-grade gastric mucosa-associated lymphoid tissue lymphoma after erad-
ication of Helicobacter pylori: possible association with p16 hypermethylation. J Gastroenterol 2002;37(1):17e22.
[58] Kaneko Y, Sakurai S, Hironaka M, Sato S, Oguni S, Sakuma Y, et al. Distinct methylated profiles in Helicobacter pylori dependent and independent
gastric MALT lymphomas. Gut 2003;52(5):641e6.
[59] Go JH. Methylation analysis of cyclin-dependent kinase inhibitor genes in primary gastrointestinal lymphomas. Mod Pathol 2003;16(8):752e5.
[60] Koyama M, Oka T, Ouchida M, Nakatani Y, Nishiuchi R, Yoshino T, et al. Activated proliferation of B-cell lymphomas/leukemias with the SHP1 gene
silencing by aberrant CpG methylation. Lab Investig 2003;83(12):1849e58.
[61] Takino H, Okabe M, Li C, Ohshima K, Yoshino T, Nakamura S, et al. p16/INK4a gene methylation is a frequent finding in pulmonary MALT lymphomas at
diagnosis. Mod Pathol 2005;18(9):1187e92.
[62] Min KO, Seo EJ, Kwon HJ, Lee EJ, Kim WI, Kang CS, et al. Methylation of p16(INK4A) and p57(KIP2) are involved in the development and progression of
gastric MALT lymphomas. Mod Pathol 2006;19(1):141e8.
[63] Huang Q, Su X, Ai L, Li M, Fan CY, Weiss LM. Promoter hypermethylation of multiple genes in gastric lymphoma. Leuk Lymphoma 2007;48(10):
1988e96.
[64] Park S, Kim KM, Kim JJ, Lee JH, Rhee JC, Ko YH. Methylation of p16INK4A and mitotic arrest defective protein 2 (MAD2) genes in gastric marginal-zone
B-cell lymphomas. Acta Haematol 2008;120(4):217e24.
[65] Toso A, Aluffi P, Capello D, Conconi A, Gaidano G, Pia F. Clinical and molecular features of mucosa-associated lymphoid tissue (MALT) lymphomas of
salivary glands. Head Neck 2009;31(9):1181e7.
[66] Sinn DH, Kim YH, Lee EJ, Ko YH, Kim KM. Methylation and API2/MALT1 fusion in colorectal extranodal marginal zone lymphoma. Mod Pathol 2009;
22(2):314e20.
[67] Kondo T, Oka T, Sato H, Shinnou Y, Washio K, Takano M, et al. Accumulation of aberrant CpG hypermethylation by Helicobacter pylori infection
promotes development and progression of gastric MALT lymphoma. Int J Oncol 2009;35(3):547e57.
[68] Chanudet E, Huang Y, Ichimura K, Dong G, Hamoudi RA, Radford J, et al. A20 is targeted by promoter methylation, deletion and inactivating mutation
in MALT lymphoma. Leukemia 2010;24(2):483e7.
[69] Choung HK, Kim YA, Lee MJ, Kim N, Khwarg SI. Multigene methylation analysis of ocular adnexal MALT lymphoma and their relationship to Chla-
mydophila psittaci infection and clinical characteristics in South Korea. Investig Ophthalmol Vis Sci 2012;53(4):1928e35.
[70] Takino H, Li C, Yamada S, Sato F, Masaki A, Fujiyoshi Y, et al. Thymic extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue: a
gene methylation study. Leuk Lymphoma 2013;54(8):1742e6.
[71] Lee MJ, Min BJ, Choung HK, Kim N, Kim YA, Khwarg SI. Genome-wide DNA methylation profiles according to Chlamydophila psittaci infection and the
response to doxycycline treatment in ocular adnexal lymphoma. Mol Vis 2014;20:1037e47.
[72] Esteller M. Profiling aberrant DNA methylation in hematologic neoplasms: a view from the tip of the iceberg. Clin Immunol 2003;109(1):80e8.
[73] Takino H, Li C, Hu S, Kuo TT, Geissinger E, Muller-Hermelink HK, et al. Primary cutaneous marginal zone B-cell lymphoma: a molecular and clini-
copathological study of cases from Asia, Germany, and the United States. Mod Pathol 2008;21(12):1517e26.
[74] Matsusaka K, Funata S, Fukayama M, Kaneda A. DNA methylation in gastric cancer, related to Helicobacter pylori and Epstein-Barr virus. World J
Gastroenterol 2014;20(14):3916e26.
[75] Zucca E, Bertoni F, Vannata B, Cavalli F. Emerging role of infectious etiologies in the pathogenesis of marginal zone B-cell lymphomas. Clin Cancer Res
2014;20(20):5207e16.
 A.J. Arribas, F. Bertoni / Best Practice & Research Clinical Haematology 30 (2017) 24e31 31

[76] Novak U, Rinaldi A, Kwee I, Nandula SV, Rancoita PMV, Compagno M, et al. The NF-ΚB negative regulator TNFAIP3 (A20) is commonly inactivated by
somatic mutations and genomic deletions in marginal zone B-cell lymphomas. Blood 2009;113(20):4918e21.
[77] Arribas AJ, Rinaldi A, Mensah AA, Kwee I, Cascione L, Robles EF, et al. DNA methylation profiling identifies two splenic marginal zone lymphoma
subgroups with different clinical and genetic features. Blood 2015;125(12):1922e31.
[78] Ansell SM. DNA methylation in lymphoma: an opportunity? Blood 2015;125(12):1848e9.
[79] Arribas AJ, Gaudio E, Arcaini L, Stathis A, Zucca E, Rossi D, et al. Targeting the epigenome and the cell signaling as novel therapeutic approaches for
splenic marginal zone lymphoma. Blood 2016. poster, Abstract (#4186).
[80] Watkins AJ, Hamoudi RA, Zeng N, Yan Q, Huang Y, Liu H, et al. An integrated genomic and expression analysis of 7q deletion in splenic marginal zone
lymphoma. PLoS One 2012;7(9):e44997.