Differential expression of NF-kappaB target genes in MALT lymphoma with and without chromosome translocation: insights into molecular mechanism.

Abstract

Mucosa-associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH and t(14;18)(q32;q21)/IGH-MALT1, which commonly activate the nuclear factor (NF)-kappaB pathway. Gastric MALT lymphomas harboring such translocations usually do not respond to Helicobacter pylori eradication, while most of those without translocation can be cured by antibiotics. To understand the molecular mechanism of these different MALT lymphoma subgroups, we performed gene expression profiling analysis of 21 MALT lymphomas (13 translocation-positive, 8 translocation-negative). Gene set enrichment analysis (GSEA) of the NF-kappaB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions have shown that translocation-positive MALT lymphomas are characterized by an enhanced expression of NF-kappaB target genes, particularly toll like receptor (TLR)6, chemokine, CC motif, receptor (CCR)2, cluster of differentiation (CD)69 and B-cell CLL/lymphoma (BCL)2, while translocation-negative cases were featured by active inflammatory and immune responses, such as interleukin-8, CD86, CD28 and inducible T-cell costimulator (ICOS). Separate analyses of the genes differentially expressed between translocation-positive and -negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. Finally, expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10-mediated NF-kappaB activation in vitro. Our findings provide novel insights into the molecular mechanism of MALT lymphomas with and without translocation, potentially explaining their different clinical behaviors.

Full text

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ORIGINAL ARTICLE
Differential expression of NF-jB target genes in MALT lymphoma with and without
chromosome translocation: insights into molecular mechanism
RA Hamoudi1, A Appert1, H Ye1, A Ruskone-Fourmestraux2,3, B Streubel4, A Chott4, M Raderer5, L Gong1, I Wlodarska6,
C De Wolf-Peeters7, KA MacLennan8, L de Leval9, PG Isaacson10and M-Q Du1
1Department of Pathology, University of Cambridge, Cambridge, UK;2Service de Gastro-enterologie, Ho ˆpital St Antoine, APHP,
Paris, France;3GELD (Groupe d’Etude des Lymphomes Digestifs), Paris, France;4Department of Pathology, Medical University of
Vienna, Vienna, Austria;5Department of Medicine I, University Hospital Vienna, Vienna, Austria;6Center for Human Genetics,
Katholieke Universiteit Leuven, Leuven, Belgium;7Department of Morphology and Molecular Pathology, University of Leuven,
Leuven, Belgium;8Molecular Medicine Unit, St James’s University Hospital, University of Leeds, Leeds, UK;9Department of
Pathology, CHU Sart Tilman, Lie `ge, Belgium and10Department of Histopathology, University College London, London, UK
Mucosa-associated lymphoid tissue (MALT) lymphoma is
characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/
BCL10-IGH and t(14;18)(q32;q21)/IGH-MALT1, which commonly
activate the nuclear factor (NF)-jB pathway. Gastric MALT
lymphomas harboring such translocations usually do not
respond to Helicobacter pylori eradication, while most of those
without translocation can be cured by antibiotics. To under-
stand the molecular mechanism of these different MALT
lymphoma subgroups, we performed gene expression profiling
analysis of 21 MALT lymphomas (13 translocation-positive,
8 translocation-negative). Gene set enrichment analysis (GSEA)
of the NF-jB target genes and 4394 additional gene sets
covering various cellular pathways, biological processes and
molecular functions have shown that translocation-positive
MALT lymphomas are characterized by an enhanced expression
of NF-jB target genes, particularly toll like receptor (TLR)6,
chemokine, CC motif, receptor (CCR)2, cluster of differentiation
(CD)69 and B-cell CLL/lymphoma (BCL)2, while translocation-
negative cases were featured by active inflammatory and
immune responses, such as interleukin-8, CD86, CD28 and
inducible T-cell costimulator (ICOS). Separate analyses of the
genes differentially expressed between translocation-positive
and -negative cases and measurement of gene ontology term in
these differentially expressed genes by hypergeometric test
reinforced the above findings by GSEA. Finally, expression of
TLR6, in the presence of TLR2, enhanced both API2-MALT1 and
BCL10-mediated NF-jB activation in vitro. Our findings provide
novel insights into the molecular mechanism of MALT lympho-
mas with and without translocation, potentially explaining their
different clinical behaviors.
Leukemia (2010) 24, 1487–1497; doi:10.1038/leu.2010.118;
published online 3 June 2010
Keywords: MALT lymphoma; gene expression profiling; NF-kB
activation; chromosome translocation
Introduction
Extranodal marginal zone
associated lymphoid tissue (MALT) originates from the MALT
acquired as a result of chronic inflammatory or autoimmune
disorders.1The etiological factors underlying these chronic
inflammatory disorders have a pivotal role in MALT lympho-
magenesis. This is best exemplified by the causative role of
B-cell lymphoma of mucosa-
Helicobacter pylori infection in gastric MALT lymphoma as
shown by the compelling evidence from the epidemiological,
laboratory and particularly clinical studies, which show long-
term complete remission of the lymphoma following H. pylori
eradication in B70% of cases.1In spite of this, the molecular
mechanisms underlying the lymphoma development are not
fully understood. Stimulations of antigen receptor by autoanti-
gen, and co-stimulatory molecule CD40 by H. pylori-specific
T cells are believed to have an important role. Recent studies
on MALT lymphoma-associated chromosome translocations
provide further insights into its molecular pathogenesis.
t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH
and t(14;18)(q32;q21)/IGH-MALT1 are specifically associated
with MALT lymphoma albeit occurring at considerately variable
frequencies in different anatomic sites.2–5Although these
translocations involve different oncogenes, molecularly their
encoded products commonly activate the canonical nuclear
factor (NF)-kB pathway,6–8accounting for their critical role in
lymphomagenesis. Nonetheless, overexpression of these onco-
genes alone is insufficient for malignant transformation as both
Em-API2-MALT1 and Em-BCL10 transgenic mice developed
splenic marginal zone hyperplasia, but not lymphoma.9,10Thus,
other molecular events are required to cooperate with these
chromosome translocations in MALT lymphoma development.
The above chromosomal translocations are always mutually
exclusive and t(11;18), the most frequent translocation in MALT
lymphoma, occurs often as the sole cytogenetic abnormality.11
Several studies suggest that there is a potential cooperation
between MALT lymphoma-associated oncogenic products and
immunological stimuli in lymphomagenesis. In the Em-API2-
MALT1 transgenic mice, immunization with the Freund0s
complete adjuvant led to development of a splenic marginal
zone lymphoma-like hyperplasia.12In vitro assay showed that
CD40 stimulation enhanced both API2-MALT1 and MALT1-
induced NF-kB activation.13However, the extent of potential
cooperation between MALT lymphoma-associated oncogenic
products and immune surface receptor signaling is unknown.
In spite of the presence of a potential overlap in the molecular
mechanism of MALT lymphoma with and without translocation
as discussed above, there are important differences in the
clinical and histological presentations between these different
subgroups. Clinically, gastric MALT lymphomas with t(11;18)
or t(1;14) are significantly associated with advanced stages
and resistance to H. pylori eradication.14,15Histologically,
t(11;18)-positive MALT lymphomas seem to be more mono-
tonous, lacking apparent transformed blasts.16These distinct
Received 4 November 2009; revised 2 March 2010; accepted 1 April
2010; published online 3 June 2010
Correspondence:
Histopathology, Department of Pathology, University of Cambridge,
Level 3 Lab Block, Box 231, Addenbrooke’s Hospital, Hills Road,
Cambridge, CB2 2QQ, UK.
E-mail: mqd20@cam.ac.uk
Professor M-QDu,Division ofMolecular
Leukemia (2010) 24, 1487–1497
& 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10
www.nature.com/leu

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clinico-pathological characteristics may indicate important
differences in molecular mechanisms between MALT lymphomas
with and without translocation. To analyze this and understand
further the molecular mechanism of MALT lymphoma, we
studied the transcriptional profiles of a well-characterized series
of cases with different translocation status and further validated
the genes identified and the hypothesis generated.
Materials and methods
Patient materials
Fresh frozen tissues from 24 well-characterized MALT lymphomas
(Supplementary Table S1), 7 follicular lymphoma (FL) and 7
mantle cell lymphoma (MCL) were used for gene expression
microarray analysis. The MALT lymphomas were nine positive
for t(11;18)/API2-MALT1 (eight gastric and one pulmonary),
four positive for t(1;14)/BCL10-IGH or t(1;2)/BCL10-IGk (three
gastric and one pulmonary), two positive for t(14;18)/IGH-MALT1
(one hepatic and one ocular adnexal) and nine gastric cases
negative for all known MALT lymphoma-associated trans-
locations. The percentage of tumor cells was estimated on
hematoxylin and eosin stained slides and crude microdissection
was performed to ensure that at least 70% tumor cells was used
for expression microarray analysis.
In addition, 73 cases of MALT lymphoma, including 18
positive for t(11;18), 8 positive for t(1;14), 9 positive for t(14;18)
and 38 negative for these translocations, were used for
validation of the expression microarray findings. The use of
archival tissues for research was approved by the local research
ethics committees of the authors’ institutions.
Gene expression microarray
RNA extraction, synthesis of labeled complementary RNA by
in vitro transcription and hybridization to Affymetrix (Affymetrix
UK Ltd., High Wycombe, UK) GeneChip HG-U133A (MALT
lymphoma) or Affymetrix H133 plus 2 (FL and MCL), quality
control analysis, microarray data normalization and nonspecific
filtering are detailed in Supplementary Methods. All microarray
data have been deposited with Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo/, GSE18736).
Unsupervised clustering
This was performed using Pearson’s correlation coefficient and
average linkage as the similarity measure and clustering
algorithm respectively within Genespring GX 7.3.1. Separate
clustering was performed among all MALT lymphoma, FL and
MCL cases and also within the MALT lymphoma group.
Gene set enrichment analysis (GSEA)
GSEA was used to identify gene sets differentially regulated
between MALT lymphoma with (13 cases) and without (8 cases)
chromosome translocation.17
identifies the gene set showing either uniformly up- or down-
regulation, for the gene sets showing both up- and down-
regulated genes, absolute GSEA was additionally performed.18A
total of 4395 gene sets were analyzed (Supplementary Table S2)
and they included (1) NF-kB target genes, collated from online
database, published works and careful bioinformatic search
(Supplementary Table S3); (2) biological pathways involved in
inflammatory and immune responses from human immunome
database,19gene ontology (GO) and ingenuity and (3) gene sets
from Molecular Signature database. The GSEA results were
As the original GSEA only
ranked according to the nominal P-value (o0.05) and false
discovery rate (p0.25) as described previously.17
For the gene sets differentially regulated between MALT
lymphoma with and without translocation, leading edge
analysis was performed to identify the biologically important
gene subset.17When generating gene sets, for each sample, only
the maximum expression value of the multi-probes for a given
gene was used for GSEA as described previously.17
Analysis of differential gene expression in MALT
lymphomas with and without translocation
For identification of differentially expressed genes between
MATL lymphoma with and without translocation, the MAS5
normalized and filtered data set was used as suggested
previously.20
The genes differentially expressed (one-way
analysis of variance test, Po0.05) between translocation-
positive and -negative MALT lymphomas were identified, and
those showing 42.5-fold differences were selected for func-
tional annotation using GO.
Functional annotation using GO
To assess the biological implication of differential gene
expression in MALT lymphomas with and without translocation,
we measured the representation of GO terms (association of
gene products with their related biological processes and
molecular functions) in the above differentially expressed genes
using Genespring and hypergeometric tests provided in the R
package (GOstats, version 2.8.0, http://www.bioconductor.org).
This allowed us to examine whether any GO term was over- or
under-represented as compared with what can occur by chance.
Independent analyses of GO categories were performed for
overexpressed genes in both translocation-positive and -
negative MALT lymphoma.
Quantitative reverse-transcription PCR (qRT-PCR),
immunohistochemistry and western blot analysis
Pleaseseedescription in
(Supplementary Table S4–S5).
the SupplementaryMethods
NF-kB reporter assay
The potential cooperation between BCL10 (or API2-MALT1) and
TLR6 expression in NF-kB activation was analyzed in vitro using
a Luciferase Reporter Assay and the experimental details are
given in the Supplementary Methods.
Results
Transcriptional profiling defines MALT lymphoma as
a distinct entity
The microarray data from 21 MALT lymphomas (13 translocation-
positive and 8 translocation-negative), 5 FL and 7 MCL passed
the microarray hybridization quality control and were further
analyzed and presented below (Supplementary Table S6). The
standard normalization and filtering across all these cases
yielded a set of 2629 probes. As expected, CD10 and BCL6
were found most highly expressed in FL, CCND1 most highly
expressed in MCL, and MALT1 most highly expressed in MALT
lymphoma with t(14;18) or t(11;18) (Supplementary Figure S1).
Unsupervisedhierarchical clustering
lymphomas were clustered as a single branch, irrespective of
their origin from different anatomic sites. Within the MALT
showed that MALT
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lymphoma group, translocation-positive cases were inter-
mingled with translocation-negative cases (Figure 1), indi-
cating that the translocation status did not have major effect
on the hierarchical clustering. We also repeated the unsuper-
vised hierarchical clustering analysis exclusively on MALT
lymphoma cases using a set of 6893 variant probes derived
from U133A and very similar results were found (Supplementary
Figure S2).
Differential expression of NF-kB target genes in MALT
lymphoma with and without chromosome translocation
As expected, the absolute GSEA revealed that a subset of the
NF-kB target genes was over-represented in the translocation-
positive MALT lymphomas, whereas another subset was
enriched in translocation-negative cases (P¼0.011, false
discovery rate¼0.005, Figure 2a, Supplementary Table S7).
Leading edge analysis showed that 19 core genes accounted for
the significant enrichment in translocation-positive cases and
the top 10 genes included CCR2A, BCL2, CD69, TLR6, TFEC,
IRF4, PRDM2, REL, CCR7 and CCR5. Whereas, 34 core genes
underscored the significant enrichment in translocation-negative
cases and the top 10 genes were PTGS2, PLAU, NR4A3, PTGIS,
IL8, CD86, CCL2, CCL11, CXCL5 and CXCL1.
NF-kB target genes potentially underpins the differential
representation of significant gene sets between MALT
lymphomas with and without chromosome
translocation
To gain further insights into the potential difference in molecular
mechanismsbetweentranslocation-positive
MALT lymphomas, we performed GSEA, in which indicated
absolute GSEA, on 4394 gene sets covering various cellular
pathways, biological processes and molecular functions. A total
of 33 gene sets (not including those with very general term
or those containing o20 genes) were differentially over-
represented between MALT lymphomas with and without
translocation (Po0.05, false discovery rateo0.20, Table 1).17
As there was a considerable overlap among the gene sets
associated with the related cellular pathways or biological
processes, they were grouped according to their involvement
in theNF-kBactivationpathway,
responses, chemokine and cell migration, G protein-coupled
receptor(GPR)signalingand
(Table 1). Leading edge analysis was performed to identify the
core subset genes that underscored the significant enrichment
and were thus most likely biologically important. Interestingly,
the NF-kB target genes were frequently presented in each of
these core subset genes, often on top of the list (Supplementary
and -negative
inflammation/immune
cell proliferation/apoptosis
Response to external biotic
stimulus such as pest, pathogen
or parasite and cellular defense
response
Cell cycle, mitosis and
regulation of progression
through the cell cycle
Immune cell activation
3X
1X
0.1X
t(1;14)t(14;18)t(11;18) NEG
FL
MCL
Figure 1
probes were obtained and used for unsupervised hierarchical clustering analysis. All MALT lymphomas are clustered as single branch, clearly
separated from both FL and MCL. Nonetheless, within MALT lymphoma group, cases with and without chromosome translocation are
intermingled together. The gene tree was ordered according to biological processes defined by GO, and the gene clusters enriched for a particular
biological process in a lymphoma subtype as shown by hypergeometric testing are indicated. The gene sets for cell cycle (GO:7049) and regulation
of progression through cell cycle (GO:74) are highly enriched in MCL, whereas those for immune cell activation (GO:45321, GO:46649) are
enriched in FL. The gene sets for immune response to biotic stimulus (GO:9607), external biotic stimulus such as pest, pathogen or parasite
(GO:9613) and cellular defense response (GO:6952) are highly enriched in MALT lymphoma. The chromosome translocation status of MALT
lymphoma, FL and MCL are indicated by different color scheme. On the heatmap, red represents upregulated genes and green downregulated
genes, with the scale showed at the bottom of the figure. FL, follicular lymphoma; MCL, mantle cell lymphoma; NEG, translocation-negative MALT
lymphoma.
MALT lymphoma shows distinct gene expression profiles from FL and MCL. After standard normalization and filtering, a set of 2629
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Tables S8–S16, Supplementary Figure S3). Figure 2b shows
the results of GSEA of immune response genes (GO:6955)
with the top 15 leading edge core genes indicated in the
heatmap illustration. Several NF-kB targets such as CCR2,
BCL2, TLR6 and IRF4 were enriched in translocation-positive
MALT lymphoma, whereas IL8, CD86 CCL2 and ICOS
wereover-representedin
(Supplementary Table S12).
translocation-negativecases
Differential gene expression between MALT lymphomas
with and without chromosome translocation
Using one-way analysis of variance test (Po0.05) and a
2.5-folds change as the threshold, we identified 26 and
62 genes significantly overexpressed in translocation-positive
andtranslocation-negative MALT
(Supplementary Table S17). To assess the biological implication
of this differential gene expression in MALT lymphoma with and
without chromosome translocation, we measured the represen-
tation of GO terms in the above gene sets using hypergeometric
tests. Among the genes overexpressed in translocation-positive
MALT lymphoma, the GO terms assocaited with NF-kB
pathway activation, chemokine/GPR signaling, and antigen
presentation were significantly over-represented (Supplementary
lymphoma respectively
Table S18). Although among the genes overexpressed in
translocation-negative MALT lymphoma, the GO terms related
to immune/defense response were significantly over-represented
(Supplementary Table S18). These findings from analysis of
differentially expressed genes between MALT lymphomas with
and without translocation reinforce the above observations
by GSEA.
Validation of gene expression by qRT-PCR and
immunohistochemistry
To confirm the differential expression of the key candidate genes
between MALT lymphoma with and without translocation
identified by the above microaray analyses, we analyzed the
expression of MALT1, BCL10, TLR6, CD69, CCR2A, CCR5,
CD86andNR4A3byqRT-PCR
tumor cells from formalin-fixed paraffin-embedded tissue and
wherein possible by immunohistochemistry in a total of 58 cases
including 16 used in gene expression profiling. As expected,
MALT1 was most highly expressed in cases with t(14;18)/IGH-
MALT1 and BCL10 was highest expressed in those with t(1;14)
(Supplementary Figure S4). In keeping with the expression
microarray data, TLR6, CD69 and CCR2A were highly
expressed in t(1;14) or t(11;18)-positive MALT lymphomas and
ofthemicrodissected
trans -vetrans +ve
Running enrichment score
(No. of genes: 5242 in list,
147 in gene set)
trans -vetrans +ve
(No. of genes: 5242 in list,
97 in gene set)
Running enrichment score
NF-kB target genes
Immune response GO:6955
5000
4000
3000
20001000 0
50004000 30002000 10000
-0.8
-0.6
-0.4
-0.2
0.0
0.2
-0.8
-0.6
-0.4
-0.2
0.0
0.2
a
b
Figure 2
translocation. Left panel shows the distribution of NF-kB target genes or immune response genes according to their rank position. Right panel
shows heatmap illustration of their expression between MALT lymphoma with and without chromosome translocation. The top 15 leading edge
core genes are shown. Trans –ve: translocation-negative MALT lymphoma; trans þve: translocation-positive MALT lymphoma.
GSEA of NF-kB target genes (a) and immune response genes (GO:6955) (b) in MALT lymphomas with and without chromosome
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Table 1
Gene sets differentially over-represented between MALT lymphoma with and without chromosome translocation
Gene sets
Source
SIZE
ES
NES
NOM
P-val
FDR
q-val
Tag
%
Gene
%
Leading edge
core set genes
NF-kB related
NF-kB target
genes
Supplementary Table S3for details
147
0.44
1.64
0.0109
0.0050
0.361
0.250
Supplementary Table S7
Positive regulation of IKK NF-kB cascade
GO:43123
43
0.49
1.80
0.0041
0.0754
0.395
0.209
Supplementary Table S8
Regulation of IKK NF-kB cascade
GO:43122
40
0.50
1.68
0.0148
0.1235
0.400
0.206
Inflamation and immune response
Response to chemical stimulus
GO:42221
118
0.42
1.64
0.0000
0.1388
0.475
0.356
Supplementary Table S9
Defense response
GO:6952
110
0.43
1.62
0.0079
0.1449
0.382
0.223
Supplementary Table S10
B-cell activation
GO:42113
21
0.58
1.57
0.0183
0.1661
0.381
0.080
Innate immune response
GO:45087
42
0.45
1.55
0.0371
0.1503
0.548
0.359
Supplementary Table S11
Immune response
GO:6955
97
0.44
1.50
0.0431
0.1633
0.433
0.299
Supplementary Table S12
Chemokine and cell migration
Cation homeostasis
GO:55080
47
0.47
1.66
0.0022
0.1280
0.362
0.193
Supplementary Table S13
Cellular cation homeostasis
GO:30003
46
0.46
1.63
0.0044
0.1376
0.348
0.193
Locomotory behavior
GO:7626
46
0.52
1.64
0.0166
0.1432
0.370
0.125
Supplementary Table S14
GPR signaling and MAPK pathway
Peptide GPCRs
MSD-C2 (Supplementary Table S2)
20
0.71
2.07
0.0000
0.0096
0.550
0.153
Supplementary Table S15
GPCRDdb class A rhodopsin like
MSD-C2 (Supplementary Table S2)
38
0.57
2.08
0.0000
0.0100
0.421
0.163
Cyclic nucleotide mediated signaling
GO:19935
26
0.55
1.83
0.0000
0.0379
0.308
0.134
G protein signaling coupled to cyclic nucleotide second messenger
GO:7187
26
0.55
1.83
0.0000
0.0379
0.308
0.134
Regulation of kinase activity
GO:43549
72
0.43
1.85
0.0000
0.0744
0.333
0.186
Regulation of protein kinase activity
GO:45859
71
0.43
1.86
0.0000
0.0838
0.338
0.186
Negative regulation of catalytic activity
GO:43086
29
0.49
1.86
0.0000
0.1257
0.483
0.275
Positive regulation of catalytic activity
GO:43085
66
0.40
1.74
0.0021
0.0876
0.273
0.178
Regulation of MAPK activity
GO:43405
34
0.48
1.84
0.0024
0.0623
0.500
0.303
Supplementary Table S16
G protein coupled receptor protein signaling pathway
GO:7186
96
0.42
1.63
0.0041
0.1337
0.302
0.185
G protein signaling coupled to camp nucleotide second messenger
GO:7188
19
0.55
1.70
0.0043
0.1108
0.316
0.134
Positive regulation of signal transduction
GO:9967
51
0.43
1.63
0.0143
0.1399
0.471
0.348
Proliferation and apoptosis
Growth
GO:40007
24
0.52
1.74
0.0022
0.0930
0.458
0.255
Regulation of growth
GO:40008
22
0.51
1.64
0.0044
0.1409
0.455
0.255
Regulation of cell growth
GO:0001558
21
0.50
1.62
0.0065
0.1415
0.429
0.255
Anti-apoptosis
GO:6916
70
0.38
1.50
0.0397
0.1514
0.300
0.218
Others
B-cell lymphoma
GeneGo
71
0.43
1.53
0.0489
0.1516
0.507
0.340
Regulation of transferase activity
GO:51338
74
0.42
1.84
0.0000
0.0519
0.432
0.303
Regulation of translation
GO:6417
26
0.52
1.77
0.0000
0.0699
0.615
0.364
Lian myeloid diff receptors
MSD-C2 (Supplementary Table S2)
17
0.71
1.87
0.0000
0.1504
0.647
0.156
Zhan MM CD138 CD2 BS rest
MSD-C2 (Supplementary Table S2)
24
0.57
1.83
0.0000
0.1947
0.625
0.267
Behavior
GO:7610
57
0.51
1.69
0.0043
0.1138
0.333
0.125
Ion homeostasis
GO:50801
50
0.45
1.59
0.0174
0.1728
0.340
0.193
Abbreviations: ES, enrichment score; FDR, false dicovery rate; Gene %, the percentage of genes in the gene list before (for positive ES) of after (for negative ES) the peak in the running enrichment
score; GO, gene ontology; GPCRs, G protein-coupled receptors; GPR, G protein-coupled receptor; MAPK, mitogen-activated protein kinase; MSD, molecular signature database (http://
www.broadinstitute.org/gsea/msigdb/index.jsp); NES, normalised ES; NF, nuclear factor; NOM, nominal; Tag %, the percentage of gene tags before (for positive ES) of after (for negative ES) the peak in
the running enrichment score.
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CCR5 was highly expressed in t(1;14)-positive cases in
comparison with translocation-negative cases (Supplementary
Figure S4). Conversely, CD86 and NR4A3 were significantly
highly expressed in translocation-negative MALT lymphomas
(Supplementary Figure S4).
In keeping with the above qRT-PCR data, immunohisto-
chemistry showed that most translocation-positive MALT
lymphomas showed strong to moderate homogeneous BCL2
and CD69 staining in 470% tumor cells, often in most
tumor cells, whereas majority of translocation-negative cases
showed heterogeneous staining in 30–70% cells or a negative
result (Figure 3). Similarly, western blot analyses showed
that TLR6 was highly expressed in translocation-positive
MALT lymphoma in comparison with the translocation-negative
t(11;18) positive MALT lymphomatranslocation negative MALT lymphoma
M1 M2 M3 M4M5 M6M7 M8M9M10 M11 M12M13 M14FLJurkat
1.10 0.76 0.80 0.81 0.63 0.67 0.02 0.05 0.05 0.07 0.06 0.260.01 0.390.790.27
t(11;18)
positive
Trans -ve
0%
25%
50%
75%
CD86 BCL2CD69
FC
100%
N=28
Trans+ve
N=26
Tans-ve
BCL2
N=29
Trans+ve
N=41
Tans-ve
CD69
N=33
Trans+ve
N=26
Tans-ve
CD86
Negative
Weak staining in 30-70% cells
Strong –moderate staining in 30-70%
Weak staining in 70% cells
Strong –moderate staining in 70%
TLR6/Actin
TLR6
β-actin
a
b
Figure 3
examples of BCL2, CD69 and CD86 immunohistochemistry in MALT lymphomas with and without chromosome translocation. Original
magnification ?400 for all panels. Lower panel summaries BCL2, CD69 and CD86 immunohistochemical results in MALT lymphoma with and
without chromosome translocation. BCL2 and CD69 are more strongly and homogeneously expressed in translocation-positive than translocation-
negative MALT lymphoma (P¼6.9?10?5, P¼2.2?10?4respectively by Fisher’s exact test), whereas CD86 is more strongly expressed in
translocation-negative than translocation-positive MALT lymphoma (P¼6.4?10?7by Fisher’s exact test). FC, follicle center; Transþve,
translocation-positive; Trans ?ve, translocation-negative. (b) Western blot analysis shows that TLR6 is highly expressed in translocation-positive
MALT lymphoma, but at low levels in translocation-negative cases. M, MALT lymphoma; FL, follicular lymphoma.
BCL2, CD69, CD86 and TLR6 protein expression in MALT lymphomas with and without chromosomal translocation. (a) Top panel:
NF-jB activation in MALT lymphoma
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cases (Figure 3). In contrast, most translocation-positive
MALT lymphomas showed no CD86 staining, whereas the
majority of translocation-negative cases showed heterogeneous
CD86 staining albeit variable in both positivity and intensity
(Figure 3).
TLR6 expression enhances NF-kB activation by BCL10
and API2-MALT1 in vitro
Among the genes highly expressed in translocation-positive
MALT lympoma, TLR6, CCR2A, CD69 and BCL2 were
particularly interesting and we selected TLR6 for further
functional investigation because overexpression of this pattern
recognition receptor may sensitize the response of tumor
cells, particularly those with translocation, to stimulation by
microbial antigens. To attest this, we performed a series of
NF-kB reporter assays in Jurkat T cells, which are known not
responding to lipopolysaccharides (LPS) stimulation, thus ideal
for analyzing TLR signaling. Expression of TLR6 alone did not
enhance BCL10 or API2-MALT1-induced NF-kB activation in
Jurkat cells even in the presence of LPS stimulation (Supple-
mentary Figure S5). TLR6 functions through the formation of
heterodimer with its family member and typically forms
heterodimer with TLR2 in responding to stimulation by bacterial
antigen.21We next analyzed whether co-expression of TLR6
and TLR2 could enhance BCL10 or API2-MALT1-mediated
NF-kB activation in the presence of LPS stimulation. As
expected, TLR6/2 co-expression, in the presence of LPS
stimulation, were synergistic with BCL10 and API2-MALT1 in
activating the NF-kB pathway (Figure 4). In contrast, there was
no cooperation between co-expression of TLR6/1 and BCL10 or
API2-MALT1 in NF-kB activation (Figure 4).
Discussion
This study showed that MALT lymphoma was characterized by
distinct expression profile in comparison with FL and MCL, in
line with the recent finding by Chng et al.22Although
unsupervised clustering analyses showed considerable overlap
in the gene expression profiles between MALT lymphomas with
and without chromosome translocation, there was important
difference in the expression of NF-kB target genes between the
two subgroups. By exhaustive GSEA of various molecular
pathways and biological processes, we also showed that the
gene sets related to inflammation, immune responses, chemo-
kine and GPR signaling are differentially over-represented
between these different subgroups. Importantly, several of these
molecular pathways or biological processes also lead to NF-kB
activation. These findings were reinforced by independent
analyses of differentially expressed genes between MALT
lymphomas with and without translocation using hypergeo-
metric tests. Our observations provide several novel insights into
the molecular mechanisms of both translocation-positive and
-negative MALT lymphomas and potentially explain their
different clinical and histological presentations.
Molecular mechanism of translocation-positive
MALT lymphoma
In comparison with translocation-negative MALT lymphoma,
GSEA and leading edge analyses revealed a common core
subset of genes that were overexpressed in translocation-
positive cases and a high proportion of them are NF-kB target
genes involving multiple related biological processes or
molecular pathways. The top examples included immune
0
10
20
30
40
BCL10AM TLR6/2 BCL10
TLR6/2
AM
TLR6/2
TLR6/1 BCL10
TLR6/1
AM
TLR6/1
BCL10-Flag
AM-Flag
TLR-Flag
β-actin
No stimulation
LPS stimulation
Fold increase of NF-κB activity
vector
Anti-Flag
Anti-actin
∗
∗
∗
Figure 4
were co-transfected with vector (pIRESpuro2) or plasmids containing BCL10, API2-MALT1 (AM), TLR6, TLR2 and TLR1 as indicated, together with
NF-kB luciferase reporter gene. The transfected cells were seeded in multiple cell plates, cultured for 20h and then treated with LPS or vehicle
alone for 6h. NF-kB activities were measured in quadruplet experiments and recorded as fold increase in the vector control. Western blot in the
lower panel shows appropriate expression of various expression constructs. *Po0.01 by Student’s t-test.
TLR6 enhances BCL10 and API2-MALT1-mediated NF-kB activation, in presence of TLR2 but not TLR1, in Jurkat T cells. Jurkat T cells
NF-jB activation in MALT lymphoma
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receptors such as TLR6, TLR7, CD69 and CD1D, and
chemokine receptor such as CCR2, CXCR4, CCR6 and CCR7,
the apoptosis inhibitor BCL2, and positive regulators of the
NF-kB pathway such as REL and molecules involved in GPR
signaling (Figure 2, Supplementary Tables S7–S16). The over-
expression of CCR2, BCL2, CD69 and TLR6 in translocation-
positive cases was further confirmed in a large cohort of MALT
lymphomas by qRT-PCR and/or immunohistochemistry/western
blot analysis. All these molecules are expected to promote
tumor cell survival and proliferation either directly or indirectly.
Among these, the overexpression of the above immune surface
receptors and chemokine receptors is particularly interesting.
TLR are critical in surveillance of microbial infection by
recognizing pathogen-associated molecular patterns such as LPS
and bacterial lipopeptides. In mouse model, it has been shown
that TLR signaling promotes marginal zone B-cell activation and
migration.23TLR6 typically forms heterodimers with TLR2 on
the cell surface to recognize bacterial antigens.21TLR2/TLR6
signaling activates not only IkappaB kinase (IKK) complex that
leads to activation of the NF-kB transcriptional factor, but also
the mitogen-activated protein kinase (MAPK) p38 and Jun
amino-terminal kinase that lead to activation of the activator
protein 1 (AP-1) transcriptional factor.24Hence, overexpression
of TLR6 in translocation-positive MALT lymphoma could
potentially augment the NF-kB activity mediated by MALT
lymphoma-associated oncogenic products and also activate the
MAPK pathways. In this study, we tested the former hypothesis
and showed indeed that expression of TLR6, in presence of
TLR2, could enhance both BCL10 and API2-MALT1-mediated
NF-kB activation in vitro and this effect was particularly
significant on LPS stimulation. A role of TLR signaling in the
pathogenesis of translocation-positive MALT lymphoma is also
suggested by the followings: (1) H. pylori infection is invariably
associated with translocation-positive gastric MALT lymphoma;
(2) H. pylori activates NF-kB through both the classical and
alternative pathway in B lymphocytes and this effect is
dependent on LPS but not cag pathogenecity island;25(3)
H. pylori-associated LPS-induced NF-kB activation requires
TLR2/TLR6 or TLR2/TLR1 complex.26Taken together, these
findings suggest that there is a potential biological cooperation
between MALT lymphoma translocation and TLR signaling in
the lymphomagenesis.
CD69, a type II transmembrane glycoprotein, is a potential
co-stimulatory receptor and may also have an immunoregula-
tory role.27Although the precise function of CD69 in B cells is
largely unknown, it is a well-described activation marker in
several cell types, and its expression is upregulated in marginal
zone B cells on TLR stimulation.23CD69 is frequently expressed
in low-grade B-cell lymphomas, and in FL, its expression is
associated with poor treatment outcome.28,29Our finding of
enriched expression of CD69 in translocation-positive MALT
lymphoma further implicates its role in lymphoma pathogenesis.
CCR are GPRs and mediate immune cells migration and their
retention in the inflammatory site. As B-cell homeostatic
chemokine receptor, CCR7, CCR6 and CXCR4 are crucial for
this homing process. For example, CCR7 has a central role in the
regulation of normal mucosal lymphocyte re-circulation and
homeostasis, particularly in the stomach,30and CXCR4 is
critical for B-cell homing to the Peyer’s patches and splenic
marginal zone.31Although the specific role of CCR2 in B-cell
trafficking and homing is unclear, it forms heterodimer with
CXCR4,32thus potentially having a role in mature B-cell homing
process. In both low-grade B-cell lymphomas and classic
Hodgkin lymphomas, CCR7 and CXCR4 overexpression were
associated with a wide lymph node spread, supporting their role
in lymphoma pathogenesis.33–35In addition to homing process,
CCR signaling may also promote cell survival and proliferation
through its activation of MAPK pathways. In this context, it is
noteworthy that GPR is also targeted by chromosomal trans-
location in MALTlymphoma.
deregulation of GPR34 expression by t(X;14)(p11;q32) in a
salivary gland MALT lymphoma.36Importantly, expression of
GPR34-induced activation of both the NF-kB and MAPK
pathways in vitro.36In keeping with these findings, our GSEA
also showed that several gene sets related to GPR signaling
and MAPK pathways were enriched in translocation-positive
MALT lymphoma.
As discussed above, several molecular pathways including
signaling through TLR, and chemokine receptor may be opera-
tional in translocation-positive MALT lymphomas and contribute
to the activation of the NF-kB pathway (Figure 5). Together with
MALT lymphoma-associated oncogenic products, they cause
relentless NF-kB activation, leading to the prolonged survival of
tumor cells even in the case of obliteration of microbe-mediated
immune responses, such as H. pylori eradication in gastric MALT
lymphoma. In this regard, it is to be noted that the apoptosis
inhibitor BCL2 was remarkably uniformly overexpressed virtually
in all tumor cells in nearly all translocation-positive cases. In
contrast, the protein was heterogeneously expressed, at a much
lower level, in tumor cells of translocation-negative cases.
In spite of the above overwhelming evidence of NF-kB
activation in translocation-positive MALT lymphoma, there was
considerable heterogeneity in the expression of NF-kB target
genes among these lymphomas. Not all translocation-positive
MALT lymphomas showed uniform overexpression of the
leading edge core set of the NF-kB target genes described
above, nor each of the translocation-negative cases showed a
complete lack of expression of these NF-kB target genes
(Figure 2, Supplementary Figure S4). This is also consistent with
the clinical response of gastric MALT lymphoma to H. pylori
eradication therapy. Although most of t(11;18)-positive gastric
MALT lymphomas do not respond to H. pylori eradication, there
are occasional cases responsive to the antibiotic treatment,37
suggesting that not all translocations have the same biological
effect. Equally, the majority of translocation-negative gastric
MALT lymphomas can be cured by H. pylori eradication, but
there are 10–20% cases that are negative for MALT1, BCL10 and
FOXP1 involved translocations, and do not respond to H. pylori
eradication,37suggesting presence of other unknown genetic
abnormalities that may also target the NF-kB pathway.
A recentstudyreported
Molecular mechanism of translocation-negative
MALT lymphoma
In contrast to translocation-positive MALT lymphoma, translo-
cation-negative cases were characterized by expression of
a strong inflammatory gene signature. GSEA and leading
edge analysis also revealed common core subset genes
involving several related biological processes or molecular
pathways, which were enriched in translocation-negative MALT
lymphoma. The top examples included proinflammatory cyto-
kines IL8 and IL1b, molecules involved in B- and T-cell
interaction such as CD86, CD28 and ICOS, several chemokine
and chemokine receptors and NR4A3 (also known as MINOR)
(Figure 2, Supplementary Tables S7, S9, S10, S12, S14).
IL8 and IL1b are the hallmark of proinflammatory cytokine
profile in response to H. pylori infection. IL8 is critical for
neutrophil infiltration and activation, whereas IL1b induces
gastrin release, inhibits acid secretion and promote apoptosis of
epithelial cells.38The finding of overexpression of these
NF-jB activation in MALT lymphoma
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proinflammatory cytokines in translocation-negative gastric MALT
lymphomas, indicates the presence of active H. pylori infection. In
keeping with this, translocation-negative gastric MALT lympho-
mas show a higher number of blast cells than translocation-
positive cases.16In addittion, a number of chemokines and
chemokine receptors were highly expressed in the translocation-
negative cases. This may reflect the trafficking and retention of
various immune cells in response to an active H. pylori infection.
Most importantly, GSEA showed enriched expression of the
surface molecules involved in B- and T-cell interaction namely
CD86, CD28 and ICOS in translocation-negative gastric MALT
lymphoma. Although residual reactive follicles may be present
and contribute to the high CD86, CD28 and ICOS expression in
translocation-negative cases, the germinal center markers CD10
and BCL6 were expressed in much low levels in MALT
lymphoma (Supplementary Figure S1), and more importantly
overexpression of CD86 in tumor cells was clearly shown
by qRT-PCR and immunohistochemistry. In line with our
finding, a previous study showed significantly higher CD86
expression in gastric MALT lymphomas that responded to
H. pylori eradication than those resisted to the therapy
(66% vs 10%).39Although the chromosome translocation status
in these cases is not available, it is most likely that the cases
responded to H. pylori were translocation-negative.14Taken
together, these findings suggest that there is an active immune
response to H. pylori infection in translocation-negative gastric
MALT lymphoma, and this most likely underscores the tumor
cell survival and expansion, and thus determines their response
to H. pylori eradication (Figure 5).
In summary, this study shows that (1) translocation-positive
MALT lymphoma is in general characterized by an enhanced
expression of NF-kB target genes, particularly CCR2, TLR6
and BCL2; (2) the oncogenic products of MALT lymphoma-
associated translocation may cooperate with signaling from
several surface receptors including TLR6 and chemokine
receptors in activation of the NF-kB pathways; and (3)
translocation-negative MALT lymphoma is featured by active
inflammatory and immune responses to H. pylori infection,
and tumor cell interaction with infiltrating T cells through
co-stimulating molecules (especially CD86/CD28) may have
an important role in their survival and clonal expansion.
Conflict of interest
The authors declare no conflict of interest.
Chromosome translocation
positive MALT lymphoma
MAPK
p50
p65
IKB
P
p100
RelB
p52
RelB
target genes
Nucleus
Canonical pathwayNon-canonical pathway
API2-MALT1
MALT1
BCL10
BCL2
CCR2
CD69
TLR6
Apoptosis
LTβR
CD69
CCR2
TLR6
p50
p65
Chromosome translocation
negative MALT lymphoma
CCL2
& others
TLR2
CD86
T cell
CD28
ICOS
↑↓
B7RP1
↑↓
lymphoma B cell
Figure 5
positive MALT lymphoma, overexpression of API2-MALT1, BCL10 and MALT1 activates the canonical NF-kB pathway, leading to enhanced
expression of the NF-kB target genes, particularly TLR6, CCR2, CD69 and BCL2. Overexpression of TLR6 may provide a further positive feedback
to the activation of the NF-kB pathway. Similar positive feedback may also be expected from the CCR2 signaling, and in addition both TLR6 and
CCR2 may trigger activation of the MAPK pathway. The pathogenic implication of enhanced CD69 expression is currently unknown.
Overexpression of BCL2 is expected to promote the tumor cell survival. In essence, the above chromosome translocations cause constitutive
NF-kB activation with expression of its target genes forming a potential positive feedback loop, and the relentless NF-kB activation, in the
case of gastric MALT lymphoma, confers its resistance to H. pylori eradication. In translocation-negative MALT lymphoma, the ongoing
inflammatory and immune responses maintain active cognate B and T cell interaction through co-stimulating molecules CD86/CD28, B7RP1/
ICOS, which are the major determinants of tumor cell survival and thus explain, in the cases of gastric MALT lymphoma, their responses to
H. pylori eradication.
Summary and hypothesis on molecular mechanism of MALT lymphoma with and without chromosomal translocation. In translocation-
NF-jB activation in MALT lymphoma
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Acknowledgements
We would like to thank Professor Ahmet Dogan and Dr Ellen
Remstein, Department of Pathology, Mayo Clinic for sharing their
pulmonary MALT lymphoma gene expression microarray data;
Dr Ian McFarlane, the Microarray CoreLab, National Institute
of Health Research, Cambridge Comprehensive Biomedical
Research Centre for his technical assistance, Dr Koichi Kuwano,
Department of Infectious Medicine, Kurume University School
of Medicine, Japan for providing TLR1, TLR2 and TLR6
expression constructs and both previous and present members
of Du lab for helpful discussion and assistance. The research
in Du lab was supported by research grants from Leukemia
Research, UK and the National Institute for Health Research
Cambridge Biomedical Research Center. BS was supported by
Grant FWF (P19346-B12). LdL is a senior research associate of the
FRS-FNRS.
Authors contribution
RAH designed the experiment, collected and analyzed the data.
AA, HY and LG contributed to the design and experimental data
collection and analysis; ARF, BS, AC, MR, IW, CDWP, KAM,
LdL and PGI provided lymphoma cases; MQD designed,
analyzed the data and wrote the paper.
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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)
NF-jB activation in MALT lymphoma
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