Histopathology 2012 DOI: 10.1111/j.1365-2559.2012.04289.

IRTA1 is selectively expressed in nodal and extranodal

marginal zone lymphomas
Brunangelo Falini, Claudio Agostinelli,1 Barbara Bigerna, Alessandra Pucciarini,
Roberta Pacini, Alessia Tabarrini, Flavio Falcinelli, Milena Piccioli,1 Marco Paulli,2
Marcello Gambacorta,3 Maurilio Ponzoni,4 Enrico Tiacci, Stefano Ascani,5 Maria Paola Martelli,
Riccardo Dalla Favera,6 Harald Stein7 & Stefano A Pileri1
Institute of Haematology, University of Perugia, Ospedale S. Maria della Misericordia, Perugia, 1Department of
Haematology and Clinical Oncology ‘L. and A. Seràgnoli’, Haemopathology Unit, University of Bologna, Policlinico S.
Orsola, Bologna, Italy, 2Department of Paediatric Sciences and Human and Hereditary Pathology, Pathologic Anatomy
Section, Pavia University, Policlinico S. Matteo, Pavia, 3Department of Laboratory Medicine, Ospedale Niguarda Ca’,
Granda, 4Pathology Unit, Department of Oncology, University Scientific Institute San Raffaele, Milan, 5Institute of
Pathology, University of Terni, Terni, Italy, 6Institute for Cancer Genetics, Columbia University, New York, NY, USA, and
7
Berlin Reference and Consultation Centre for Lymphoma and Haematopathology, Pathodiagnostik Berlin, Berlin, Germany

Date of submission 27 February 2012

Accepted for publication 28 March 2012

Falini B, Agostinelli C, Bigerna B, Pucciarini A, Pacini R, Tabarrini A, Falcinelli F, Piccioli M, Paulli M, Gambacorta M,
Ponzoni M, Tiacci E, Ascani S, Martelli M P, Dalla Favera R, Stein H & Pileri S A
(2012) Histopathology
IRTA1 is selectively expressed in nodal and extranodal marginal zone lymphomas

Aims: The aim of this study was to search for a
molecule selectively expressed by marginal zone (MZ)
lymphomas (MZLs), whose diagnosis is currently based
on morphological criteria and negativity for markers
detectable in other B-cell lymphomas.
Methods and results: Two thousand one hundred and
four peripheral lymphomas of various types were
immunostained with a monoclonal antibody against
immunoglobulin superfamily receptor translocation-
associated 1 (IRTA1), which recognizes the equivalents
of MZ in human lymphoid tissues other than spleen.
IRTA1 expression was restricted to extranodal (93%)
and nodal MZLs (73%) and to lymphomas with MZ
differentiation. Extranodal MZL cells with the strongest
IRTA1 expression were usually located adjacent to
Keywords: immunohistochemistry, IRTA1, marginal zone lymphomas, monoclonal antibodies
epithelia, mimicking the IRTA1 expression pattern of
normal and acquired mucosa-associated lymphoid tis-
sue (MALT). The cytological features, growth pattern
and IRTA1 positivity in nodal MZLs suggest they may
derive from IRTA1+ perifollicular B cells or monocytoid
B cells detectable in reactive lymph nodes. Double
immunostaining for IRTA1 ⁄ bcl-6 tracked the coloniza-
tion of B-cell follicles by MZL cells, and showed
modulation of their phenotype (e.g. acquisition of bcl-
6) during recirculation through germinal centres. MZL
cells differentiating into plasma cells usually lost IRTA1.
Conclusions: These results further expand our knowl-
edge of the biology of MZLs, and highlight IRTA1 as the
first positive marker for MZLs, enabling more accurate
diagnosis of these neoplasms.

guishes three categories of marginal zone (MZ)

Introduction
The World Health Organization (WHO) classification
of human lympho-haematopoietic neoplasms1 distin-
Address for correspondence: B Falini, Institute of Haematology,
University of Perugia, Strada Sant’Andrea delle Fratte, 06126,
Perugia, Italy. e-mail: faliniem@unipg.it
lymphoma (MZL), according to the primary site of
involvement: (i) extranodal MZL; (ii) nodal MZL; and
(iii) splenic MZL.
Criteria for the diagnosis of MZLs are mainly based
on the cytological appearance of tumour cells (e.g.
centrocytic-like or monocytoid) and their growth

 2012 Blackwell Publishing Limited.

2 B Falini et al.
pattern, e.g. formation of lympho-epithelial lesions in
extranodal tumours, or localization around reactive
follicles with expansion in the interfollicular areas in
the nodal forms.1 Because of the lack of a specific
marker for MZL,1 the immunohistological confirmation
of a morphological diagnosis of MZL is mainly based on
the demonstration that neoplastic cells lack markers
that are characteristically detectable in B-cell lympho-
mas other than MZL, e.g. bcl-6 and CD10 [both
expressed in follicular lymphoma (FL)] or CD5, CD23,
and cyclin D1, which help to define B-cell chronic
lymphocytic leukaemia (CLL) (CD5+ ⁄ CD23+ ⁄ cyclin
D1)) and mantle cell lymphoma (MCL)
(CD5+ ⁄ CD23) ⁄ cyclin D1+).1
Immunoglobulin superfamily receptor translocation-
associated 1 (IRTA1)2 is characterized by four extra-
cellular Ig-type domains and three potential ITIM
motifs in its intracellular domain.3 It belongs to a
family of five cell surface receptors named IRTA1–
IRTA53 or Fc receptor homologue 1–54,5 that are
thought to be involved in B-cell-mediated immune
responses (like Fc receptors),6 intercellular communi-
cation [like insulin receptor substrate and cell adhesion
molecule (CAM) family members],7 and cell migration
(like CAM family members).8
We previously generated a monoclonal antibody
(mAb) specifically directed against the external
portion of IRTA1,9 and demonstrated that this
molecule is selectively expressed by a subpopulation
of B cells located underneath and within the
epithelium of the tonsil and the dome epithelium
of Peyer patches, two sites that are regarded as
anatomical equivalents of the MZ.10 Moreover, we
found that, in reactive lymph nodes, the expression
of IRTA1 was restricted to a small population of B
cells located at the border of mantle cell areas and
to monocytoid B cells.9 The latter cells are usually
found in a parasinusal distribution adjacent to
subcapsular and intermediary sinuses in various
infectious and reactive lymphadenites, including
those caused by toxoplasma11,12 and several
viruses.13 The IRTA1+ cells of mucosa-associated
lymphoid tissue (MALT) and monocytoid B cells
may represent the cells of origin of extranodal and
nodal MZLs, respectively.
In order to address this issue, we investigated by
immunohistochemistry the expression of IRTA1 in
paraffin sections from 2104 cases of peripheral lym-
phoma of various categories. Evidence is provided that
the expression of IRTA1 is mainly restricted to MZLs,
especially of the extranodal and nodal types. These
findings are of obvious biological and diagnostic
relevance.
Materials and methods
mab a gainst human i rta 1
The murine mAb against IRTA1 was generated as
described previously.9 By immunohistochemistry, the
anti-IRTA1 mAb labels intraepithelial and subepithelial
MZ B cells of MALT and monocytoid B cells.9 In
western blotting, it recognizes a protein of the expected
molecular mass of 70–75 kDa.9
o t h e r an t i b o d i e s
Monoclonal antibodies (mAbs) directed against the
following antigens were used: bcl-2, CD3, CD5, CD20,
CD21, CD30, CD79a, IgD, MIB1 (Dako A ⁄ S, Glostrup,
Denmark), CD23 (Menarini Diagnostics, Wokingham,
UK), cyclin D1 (Thermo Scientific, Freemont, CA, USA),
PAX5 (Transduction Laboratories, San Diego, CA, USA),
and CD138 (Serotec, Kidlington, UK). Anti-multiple
myeloma oncogene 1 (MUM1) ⁄ interferon regulatory
factor 4 (IRF4)14 and anti-bcl-6 mAbs15 were generated
in the laboratory of one of the authors (B.F.).
tissue samp les
As B5-fixed material is not suitable for immunohisto-
chemical detection of IRTA1,9 this study was carried
out on sections from formalin-fixed paraffin-embedded
samples. We investigated 2104 lymphoma samples
representative of most categories of the 2008 WHO
classification of lympho-haemopoietic neoplasms
(Table 1). A case was regarded as positive when at
least 30% of the neoplastic cells expressed IRTA1,
according to formerly defined criteria.16 Tonsil speci-
mens were used as a positive control for the expression
of IRTA1.
single and double immunohistochemical
s t a in i ng
Following antigen retrieval (750 W for three cycles of
5 min each) in 1 mm EDTA buffer, pH 8.0,17 paraffin
sections (3–5-lm thick) were immunostained with the
immuno-alkaline phosphatase (APAAP) procedure.18
A minority of cases were tested with the peroxidase-
based EnVision method (Dako A ⁄ S). The mAb super-
natant against IRTA1 was applied at a 1:10 dilution.
Slides were then counterstained for 5 min in haemat-
oxylin and mounted in Kaiser glycerol gelatine.
Double staining for IRTA1 and bcl-6 was carried out
with a sequential procedure. Unmasking was per-
formed by heating sections in Tris ⁄ EDTA buffer
(50:2 mm, pH 9.0) in a pressure cooker in a microwave
 2012 Blackwell Publishing Ltd, Histopathology
 IRTA1 in marginal zone lymphomas 3

Table 1. Immunoglobulin superfamily receptor transloca- oven at 900 W for 3 min when at pressure. The anti-
tion-associated 1 (IRTA1) expression in 2104 lymphomas bcl-6 antibody was applied, and then revealed with the
IRTA1+ peroxidase-based EnVision technique. The IRTA1 mol-
Lymphoid tumours n cases % ecule was subsequently labelled with the APAAP
procedure.18 Slides were counterstained for 20 s in
Chronic lymphocytic 325 0* 0
leukaemia ⁄ SLL
Gill’s haematoxylin and then mounted in Kaiser
glycerol gelatine.
Lymphoplasmacytic lymphoma 30 0 0
B-cell marginal zone lymphoma fluorescence in-situ hybri d iz a tion
Splenic 21 0* 0
Tissue sections (4–6 lm thick) from two selected cases
Nodal 210 154 73 were investigated by FISH. A bcl-2 breakapart probe
Extranodal 329 307 93 (Vysis LSI bcl-2; Abbott, Abbott Park, IL, USA) and
centromere-specific probes [CEP3 (D3Z1) and CEP18
NOS 30 22 73
(D18Z1); Abbott] were applied for the detection of
Follicular lymphoma t(14;18) and trisomies 3 and 18, respectively. The cut-
Grade 1 ⁄ 2 130 0* 0 off value for the diagnosis of each probe set was the
Grade 3A 89 0* 0
mean percentage of cells with a false-positive signal
constellation plus three standard deviations, as
Grade 3B 21 0* 0 assessed on tissue from 20 reactive lymph nodes. FISH
NOS† 80 0* 0 was performed according to published standard
methods.19,20
Mantle cell lymphoma 121 0* 0
Hairy cell leukaemia 30 0 0 Results
Diffuse large B-cell lymphoma 256 69 27 The results of immunohistological analysis of 2104
Burkitt lymphoma 71 0 0 lymphoma samples are shown in Table 1 and reported
in more detail below.
Extramedullary plasmacytoma 6 0 0
Classical Hodgkin lymphoma 91 0 0 i rt a 1 is det ect ed in extranodal mzls and is
NLPHL 6 0 0 e xp re s se d a t h ig h er i n t en s it y in tu m o u r c e ll s
f or m i n g ly m p h o - e p i t h e l i a l l e s i o n s
Peripheral T-cell 160 0 0
lymphoma ⁄ NOS Because IRTA1 is selectively associated with normal
(Peyer’s patches) and acquired MALT,9 we first
Angioimmunoblastic 48 0 0
T-cell lymphoma searched for the expression of this molecule in 329
MZLs arising at various extranodal sites, including skin
Anaplastic large-cell 27 0 0 (n = 69), soft tissue (n = 14), breast (n = 14), central
lymphoma ALK+
nervous system (n = 1), eye and ocular adnexa
Anaplastic large-cell 17 0 0 (n = 33), paranasal sinus (n = 1), rhino-pharynx
lymphoma ALK) (n = 7), oral cavity (n = 3), larynx (n = 1), lung
EATL 1 0 0 (n = 24), pleura (n = 3), major salivary glands
(n = 25), oesophagus (n = 1), stomach (n = 90), small
Mycosis fungoides 5 0 0 intestine (n = 11), colon (n = 7), rectum (n = 2), liver
(n = 9), gallbladder (n = 1), kidney (n = 3), urinary
ALK, anaplastic lymphoma kinase; EATL, enteropathy-asso-
ciated T-cell lymphoma; NLPHL, nodular lymphocyte-pre- bladder (n = 1), uterus (n = 5), thymus (n = 1), and
dominant Hodgkin lymphoma; NOS, not otherwise specified; thyroid (n = 3).
SLL, small lymphocytic lymphoma. IRTA1 was expressed in at least 30% of tumour cells
*In two of 325 chronic lymphocytic leukaemias, five of 21 in 307 of 329 (93%) of extranodal MZLs. Positivity was
splenic marginal zone lymphomas, four of 121 mantle cell mainly located on the surface of tumour cells, and
lymphomas, and nine of 130 grade 1 ⁄ 2, 15 of 89 grade 3A,
eight of 21 grade 3B and 11 of 80 NOS follicular lymphomas, varied from weak to strong. Interestingly, the most
IRTA1 was expressed by a minor component of the tumour cells. intense staining for IRTA1 was observed in the
†Follicular lymphomas where grading could not be assessed. neoplastic cells giving rise to lympho-epithelial lesions;
 2012 Blackwell Publishing Ltd, Histopathology
4 B Falini et al.
those distant from the epithelium showed weak or no
expression. IRTA1+ tumour cells were small to
medium-sized, and in some samples they clustered
inside the glandular structures, producing ‘neoplastic
balls’. Representative examples of staining intensity
variability and epitheliotropism by IRTA1+ lymphoma-
tous elements in MZLs arising at different anatomical
sites are shown in Figure 1A–F.
e xp re s si o n of ir t a 1 i n n od a l an d s p le n ic m zl s
Because, in reactive lymphoid tissues, IRTA1 is selec-
tively expressed by a subset of B cells located at the
border of mantles and by monocytoid B cells9 (both
regarded as the putative cells of origin of nodal MZLs),
we next investigated its expression in 210 nodal MZLs.
IRTA1 was expressed by the tumour (at least 30%)
cells in 154 of 210 (73%) of these cases (Table 1) and,
similarly to extranodal forms, the positivity ranged
from weak to strong. In addition to tumour cells, the
antibody showed weak cross-reactivity with the endo-
thelia and sinus lining cells of the lymph nodes.
The IRTA1+ lymphoma cells frequently surrounded
reactive follicles and expanded the interfollicular areas
(Figure 2A,B,D). The morphology of IRTA1+ lymphoma
cells ranged from centrocyte-like to monocytoid
(Figure 2C,E,F). Notably, in cases with marked plasma-
cellular differentiation, tumour cells lost IRTA1 when
they acquired plasma cell morphology (Figure 3A–D).
Scattered large transformed IRTA1+ neoplastic ele-
ments, usually coexpressing CD30, were frequently
observed, especially in the interfollicular areas (not
shown).
Only two of nine nodal MZLs of the splenic type21
showed faint IRTA1 staining in a minor component of
tumour cells. A similar pattern was detected in five of
21 splenic MZLs.
i rt a 1 a n d f o l l i c u l a r co l o n i z a t i o n i n mz l s
Reactive B-cell follicles represent an important compo-
nent of both extranodal and nodal MZLs, and are
frequently replaced either partially or totally by neo-
plastic cells, thus giving rise to the pattern commonly
known as follicular colonization.10 On the basis of the
selective expression of IRTA1 in MZL cells, we used
immunohistochemistry for IRTA1 and other markers
(bcl-6, CD10, MUM1 ⁄ IRF4, and intracytoplasmic Ig) to
investigate the interaction of tumour cells with the
reactive follicles. Three main immunostaining patterns
were recognized in nodal MZLs.
Pattern 1 was characterized by the presence of B-cell
follicles that mostly contained IRTA1) ⁄ BCL6+ ⁄ CD10+
cells and were surrounded by an IRTA1+ neoplastic
population. These features are typical of nodal MZLs
with reactive non-colonized or minimally colonized
B-cell follicles (Figure 4A) and a well-preserved mesh-
work of CD21+ follicular dendritic cells (FDCs) (not
shown).
Pattern 2 was typical of nodal MZLs harbouring
reactive B-cell follicles colonized by a moderate to
marked number of IRTA1+ cells (Figure 4B). In these
cases, the colonized B-cell follicles contained numerous
bcl-6+ elements that clearly outnumbered the CD10+
cells (Figure 4C,D). Most of these elements did not
resemble centrocytes or centroblasts, but had the same
morphology as the neoplastic IRTA1+ population in the
perifollicular areas. The antibody against CD21 re-
vealed a partly preserved FDC meshwork (Figure S1).
Interestingly, double staining of two cases of nodal MZL
with these characteristics showed the presence of
IRTA1 ⁄ bcl-6 double-positive tumour elements within
the residual follicles (Figure 4E). This finding suggests
that neoplastic cells colonizing reactive follicles may
acquire a partial germinal centre cell phenotype with
expression of bcl-6 but not of CD10 (Figure 4F).
Tumour cells within B-cell follicles may eventually
undergo progressive loss of IRTA1. These two cases
were also investigated by FISH in order to exclude the
possibility of a CD10+ FL with MZ differentiation. Both
the absence of t(14;18) and presence of trisomy 3
pointed to a diagnosis of MZL.1,22
Pattern 3 of colonization was observed in cases of
nodal MZLs, where IRTA1+ tumour cells surrounded B-
cell follicles that mainly consisted of a population of
IRTA1) neoplastic cells exhibiting plasmacellular dif-
ferentiation and restriction for one of the immunoglob-
ulin light chains (Figure 5A–D). This staining pattern
most likely corresponds to follicular colonization of type
III, described by Isaacson and Norton.10 The FDC
network was largely disrupted in this setting (not
shown).
e x p r es s io n of i r t a 1 in other b-cell l ymphomas
To assess the specificity of IRTA1 expression, we
investigated 1514 peripheral lymphomas other than
MZLs. With few exceptions, the positivity for IRTA1
was restricted to MZLs. However, two of 325 CLLs, four
of 121 MCLs and 43 of 320 of FLs (mostly grade 3B)
showed expression of IRTA1 in a minor component of
the tumour cells (Table 1). Interestingly, in cases of
CLL and MCL, the IRTA1 staining corresponded to
lymphomatous elements invading the MZ of residual
follicles. In FL, it was confined to a small rim of
neoplastic cells at the periphery of pathological follicles
 2012 Blackwell Publishing Ltd, Histopathology
 IRTA1 in marginal zone lymphomas 5

A B

IRTA1

C D

IRTA1

E F

IRTA1

Figure 1. Immunoglobulin superfamily receptor translocation-associated 1 (IRTA1) in extranodal marginal zone lymphomas (MZLs). A, Gastric
MZL. Tumour cells infiltrate normal glands, giving rise to lympho-epithelial lesions (haematoxylin and eosin). B, The same case as in (A),
showing strong IRTA1 positivity of neoplastic elements. C, MZL of the thymus. The neoplastic cells infiltrate the epithelium (arrows)
(haematoxylin and eosin). D, The same case as in (C), showing that lymphoma cells expressing IRTA1 are mostly those participating in the
formation of lympho-epithelial lesions (arrow). E, MZL of the liver. Tumour cell ‘balls’ are seen within the bile ducts (arrows) (haematoxylin and
eosin). F, The same case as in (E), showing that tumour cells forming ‘balls’ are IRTA1+. B, D, F, Immuno-alkaline phosphatase technique; Gill’s
haematoxylin counterstaining.

 2012 Blackwell Publishing Ltd, Histopathology

6 B Falini et al.

A D

B E

IRTA1

C F

IRTA1 IRTA1

Figure 2. Immunoglobulin superfamily receptor translocation-associated 1 (IRTA1) in nodal marginal zone lymphomas (MZLs). A, Case 1.
Reactive follicles are separated by an interfollicular infiltrate of paler-stained neoplastic cells (asterisk) (haematoxylin and eosin). B, Neoplastic
cells in the interfollicular areas are IRTA1+ (immunoperoxidase-based EnVision technique; Gill’s haematoxylin counterstaining). C, Higher
magnification of (B). Positivity for IRTA1 is mainly located at the surface of lymphoma cells. D, Case 2. Infiltration of the lymph node by medium-
sized MZL cells (asterisk) (haematoxylin and eosin). E, Higher magnification of (D), showing tumour cells with round nuclei and watery
cytoplasm (monocytoid appearance). F, Neoplastic cells show positivity for IRTA1 (immuno-alkaline phosphatase technique; Gill’s haematoxylin
counterstaining).

 2012 Blackwell Publishing Ltd, Histopathology

 IRTA1 in marginal zone lymphomas 7

A B

IRTA1

C D

Kappa Lambda

Figure 3. Marginal zone lymphoma (MZL) cells with plasmacellular differentiation do not express immunoglobulin superfamily receptor
translocation-associated 1 (IRTA1). A, Nodal MZL with plasmacellular differentiation. B, The lymphoid component of MZL is IRTA1+, whereas
that with plasmacellular differentiation is IRTA1) (circled). C, MZL cells with plasmacellular differentiation are monotypic for cytoplasmic kappa
light chains (circled) and (D) negative for lambda light chains. B–D, Immuno-alkaline phosphatase technique; Gill’s haematoxylin
counterstaining.

reminiscent of a perifollicular MZ differentiation of the expression of IRTA1 is mostly restricted to extranodal

lymphomatous clone.
Sixty-nine of 256 (27%) diffuse large B-cell lympho-
mas (DLBCLs) were IRTA1+. No differences were
recorded between nodal and extranodal cases. Inter-
estingly, IRTA1+ DLBCLs usually showed an interfol-
licular growth pattern (Figure 6A–D) reminiscent of
that seen in typical nodal and extranodal MZLs.
Furthermore, they frequently expressed CD30 (not
shown), as observed in the large cells scattered within
low-grade MZLs.
Discussion
In this study of 2104 peripheral lymphomas, we
provide evidence that, among small B-cell lymphomas,
 2012 Blackwell Publishing Ltd, Histopathology
and nodal MZLs, whereas splenic MZLs are substan-
tially IRTA1). These findings have several biological
and diagnostic implications.
The frequent positivity of extranodal MZLs for IRTA1
is in keeping with our previous observation that the
IRTA1 molecule is expressed by the acquired MALT,9
which is regarded as the normal equivalent of MZLs.
Indeed, we provide the first immunohistological evi-
dence, based on a positive marker (IRTA1), of the
validity of the MALT and MALT lymphoma concept
first proposed by Isaacson in 1983 for low-grade B-cell
lymphomas of the gastrointestinal tract23 and later
extended to lymphomas raising from a variety of
organs,24 including unusual anatomical sites such as
kidney,25 thymus,26,27 and gallbladder.28 Interestingly,
8 B Falini et al.

A B

FL

IRTA1 IRTA1

C D

CD10 BCL6

E F

IRTA1+ IRTA1+ IRTA1+ IRTA1-

?
BCL6- BCL6- BCL6+ BCL6+

BCL6/IRTA1

Figure 4. Immunoglobulin superfamily receptor translocation-associated 1 (IRTA1) and follicular colonization in nodal marginal zone
lymphomas (MZLs). A, A case of nodal MZL showing pattern 1 of colonization. Note the presence of reactive follicles (FL) that contain rare
IRTA1+ tumour cells (arrow), indicating minimal colonization [immunoalkaline phosphatase (APAAP) technique; Gill’s haematoxylin
counterstaining]. B, A case of nodal MZL showing pattern 2 of colonization. A reactive follicle (FL) contains a marked number of IRTA1+
lymphoma cells (APAAP technique; Gill’s haematoxylin counterstaining). C, D, In the same field as (B) and at higher magnification, the bcl-6+
elements (C) in the colonized B-cell follicle definitely outnumber those that are CD10+ (D) (APAAP technique; Gill’s haematoxylin
counterstaining. E, Double staining for bcl-6 (brown) and IRTA1 (red) (sequential peroxidase-based EnVision+ method and APAAP technique;
Gill’s haematoxylin counterstaining). Most cells outside the residual follicle (FL) are IRTA1+ ⁄ bcl-6). Occasional cells within the FL are double-
positive (IRTA1+ ⁄ bcl-6+) (arrows). Cells within the FL that are bcl-6+ comprise normal residual germinal centre cells and colonizing MZL cells
that have lost IRTA1 and acquired bcl-6. F, Schematic representation of pattern 2 of follicle colonization by MZL cells. Green cells indicate the
mantle zone, and follicular dendritic cells are shown in black.

 2012 Blackwell Publishing Ltd, Histopathology

 IRTA1 in marginal zone lymphomas 9

A B

IRTA1+ IRTA1-
?

IRTA1

C D

IRF4 Ig-kappa

Figure 5. Pattern 3 of follicular colonization in nodal marginal zone lymphomas (MZLs). A, Schematic representation of reactive follicle
colonization by MZL cells with plasmacellular differentiation. Tumour cells outside the residual follicle are immunoglobulin superfamily receptor
translocation-associated 1 (IRTA1)+, whereas lymphoma cells with plasmacellular differentiation within the follicles are IRTA1). Green cells
indicate the mantle zone, and residual germinal centre cells are shown in red. B, IRTA1) reactive follicles (asterisks) are separated by an
interfollicular infiltrate of neoplastic cells showing weak to moderate positivity for IRTA1. C, Numerous neoplastic cells with plasmacellular
differentiation express MUM1 ⁄ IRF4. D, The same cells as in (C) display monotypic kappa light chain restriction. Asterisks indicate reactive
follicles. B–D, Immunoalkaline phosphatase technique; Gill’s haematoxylin counterstaining.

the IRTA1+ neoplastic cells showed the typical features
of MALT, i.e. a clear tropism for the epithelia in all
investigated anatomical sites (e.g. mucosa in MZL of
the stomach or Hassall’s corpuscles in MZL of the
thymus). The reason why the MZL cells adjacent to
epithelia express IRTA1 more strongly than those
distant from epithelia is unknown. One possible expla-
nation is that IRTA1 expression represents a marker of
area rather than of cell lineage, and that lymphoma
cells acquire the molecule once they come to the MZ.
This is, to some extent, supported by the focal IRTA1
positivity sometimes observed in cases of CLL, MCL, and
FL, which may reflect features of MZ differentiation, on
both morphological and topographic grounds.
 2012 Blackwell Publishing Ltd, Histopathology
In this study, IRTA1 expression was also recorded in
73% of nodal MZLs. This finding suggests a possible
relationship of these lymphomas with the thin rim of
IRTA1+ medium-sized lymphoid cells located at the
external part of the mantle zones of secondary follicles
of reactive lymph nodes,9 a distribution pattern that is
reminiscent of a MZ.29 Moreover, at least a proportion
of IRTA1+ nodal MZLs showed a monocytoid appear-
ance, which is in keeping with the previous observation
that IRTA1 is strongly expressed by the monocytoid B
cells in cases of reactive or infectious lymphadenitis.9
The expression of IRTA1 in both monocytoid and MZ B
cells suggests that these lymphoid subpopulations may
be somehow related, but this issue remains a matter of
10 B Falini et al.

A B

CD10

C D

IRTA1

Figure 6. Immunoglobulin superfamily receptor translocation-associated 1 (IRTA1)+ diffuse large B-cell lymphoma. A, Perifollicular growth
pattern of the tumour (Giemsa staining) (the asterisk indicates a residual B-cell follicle with a reactive germinal centre). B, Neoplastic cells are
CD10); the anti-CD10 antibody stains the residual germinal centre (asterisk). C, Higher magnification of (A) (Giemsa staining). D, Tumour cells
are IRTA1+; a similar staining pattern (not shown) is seen with the anti-CD20 antibody. B, D, Immunoalkaline phosphatase technique; Gill’s
haematoxylin counterstaining.

controversy.30,31 Lymphomas of monocytoid B cells
were first described by Sheibani et al.,32 and our finding
that they express IRTA1 further supports their present
inclusion under the common term of nodal MZL in the
WHO classification of lymphoid neoplasms.21
Notably, some other distinctive features of nodal and
extranodal MZLs can be better understood in the light of
the expression pattern of IRTA1. For example, the
finding that MZL cells undergoing prominent plasma-
cellular differentiation lose the IRTA1 molecule is in
keeping with our previous studies showing that subepi-
thelial plasma cells of the tonsil (the putative progeny of
intraepithelial B-cells)33–35 are consistently IRTA1).9
Moreover, immunostaining for IRTA1 helps to clarify
the interactions between tumour cells and reactive B-
cell follicles in both nodal and extranodal MZLs. In
particular, the phenotypic modulation of the neoplastic
cells colonizing follicles might reflect the ability of these
cells to recirculate through germinal centres by reac-
quiring expression of bcl-6 and undergoing further
rounds of somatic Ig mutations. This view is supported
by previous studies showing that IRTA1+ monocytoid B
cells residing in different areas of reactive lymph nodes
(subcapsular sinus, intermediary sinuses, and germinal
centres) can be clonally related, and that those located
in germinal centres are consistently more mutated and
selected for expression of a functional antigen receptor
than those located in the sinuses.36
In five of 21 splenic MZL cases, IRTA1 was expressed by
a minor component of the tumour cells. This finding is
consistent with the observation of occasional IRTA1+
cells in the MZ of the spleen,9 and strengthens the
 2012 Blackwell Publishing Ltd, Histopathology

hypothesis that the splenic MZ compartment is different
from the MZ in other lymphoid tissues.9,37,38 Notably, a
similar pattern of IRTA1 positivity was observed in nodal
MZLs of the splenic type,21 suggesting that these tumours
are closer to splenic MZ B cells than to the MZ and
monocytoid compartments of the normal lymph node.
Even though expression of IRTA1 was mostly
restricted to extranodal and nodal MZLs, some FLs
with MZ differentiation exhibited positivity for IRTA1
at the borders of neoplastic follicles. Moreover, occa-
sional CLL and MCL cases invading the MZ were found
to be focally IRTA1+. These findings raise the question
of whether interaction of lymphoma cells with the
microenvironment (i.e. the MZ) may be a trigger for
expression of IRTA1. They also indicate that the
immunohistological diagnosis of nodal MZL should be
based on IRTA1 expression along with the lack of other
specific B-cell lymphoma markers.
The expression of IRTA1 in a proportion of DLBCLs is
another point of interest. Notably, the distribution
pattern and the phenotypic profile of IRTA1+ neoplastic
cells in these cases were reminiscent of those observed
in typical nodal MZLs. However, whether they repre-
sent examples of transformed nodal MZL or de-novo
DLBCLs arising from monocytoid or MZ B cells remains
to be clarified.
Our findings also have important diagnostic implica-
tions. The diagnosis of extranodal MZL is accomplished
by combining clinical findings, morphological features
(especially the presence of lympho-epithelial lesions), and
immunophenotype.10 Detection of IRTA1 may be a
valuable diagnostic tool in needle biopsies, especially if
the small size of the specimen and ⁄ or the presence of
mechanical sampling artefacts hinder the evaluation of
cytology and the recognition of lympho-epithelial lesions.
Furthermore, IRTA1 facilitates the identification of the
lymphoid component in the setting of extranodal MZLs
with marked plasmacellular differentiation, allowing the
distinction from extramedullary plasmacytoma. On the
other hand, immunostaining for IRTA1 is of little value in
distinguishing extranodal MZL from atypical MZ hyper-
plasia of MALT.39,40 Under these circumstances, demon-
stration of destructive infiltrates and B-cell clonality
remains the mainstay for the differential diagnosis.
According to the 2008 WHO classification, nodal MZL
is a ‘primary nodal B-cell neoplasm that morphologically
resembles lymph nodes involved by MZL of extranodal or
splenic types, but without evidence of extranodal or
splenic disease’.21 In the absence of specific markers for
MZ B cells, distinction of nodal MZL from other small B-
cell lymphomas is based on a combination of morpho-
logical features and negativity for molecules that are
usually expressed in other lymphoma subtypes (e.g.
 2012 Blackwell Publishing Ltd, Histopathology
IRTA1 in marginal zone lymphomas 11
CD5, CD23, CD10, bcl-6, and cyclin D1).21 Thus, IRTA1
represents the first positive marker highlighting lym-
phomatous cells in nodal MZLs. Its detection becomes,
for instance, relevant in the distinction of nodal MZL
from lymphoplasmacytic lymphoma, CLL, and FL. This
holds true especially for cases with a parafollicular
pattern or diffuse growth in the absence of CD10 and
bcl-2. As for extranodal cases, IRTA1 expression is of no
relevance in distinguishing nodal MZL from reactive
nodal MZ hyperplasia.40
In conclusion, we have identified IRTA1 as the first
molecule that is significantly associated with nodal and
extranodal MZLs. These findings expand our knowledge
of the biology of MZLs, provide a novel important
diagnostic tool for MZLs, and promise to further
improve our ability to better classify these neoplasms.
Acknowledgements
We would like to thank Mrs Claudia Tibidò for her
excellent secretarial assistance and Dr Simona Righi for
her technical support. The study was supported by
grants from the Associazione Italiana per la Ricerca sul
Cancro (AIRC), BolognAIL, and Fondazione Cassa di
Risparmio in Bologna.
References
1. Swerdlow S, Campo E, Harris NL et al. World Health Organization
classification of tumours of haematopoietic and lymphoid tissues.
Geneva: IARC Press, 2008.
2. Hatzivassiliou G, Miller I, Takizawa J et al. IRTA1 and IRTA2,
novel immunoglobulin superfamily receptors expressed in B cells
and involved in chromosome 1q21 abnormalities in B cell
malignancy. Immunity 2001; 14; 277–289.
3. Miller I, Hatzivassiliou G, Cattoretti G, Mendelsohn C, Dalla-
Favera R. IRTAs: a new family of immunoglobulin-like receptors
differentially expressed in B cells. Blood 2002; 99; 2662–2669.
4. Davis RS, Wang YH, Kubagawa H, Cooper MD. Identification of a
family of Fc receptor homologs with preferential B cell expres-
sion. Proc. Natl. Acad. Sci. USA 2001; 98; 9772–9777.
5. Maltais LJ, Lovering RC, Taranin AV et al. New nomenclature for
Fc receptor-like molecules. Nat. Immunol. 2006; 7; 431–432.
6. Qiu WQ, de Bruin D, Brownstein BH, Pearse R, Ravetch JV.
Organization of the human and mouse low-affinity Fc gamma
R genes: duplication and recombination. Science 1990; 248;
732–735.
7. Ravetch JV, Lanier LL. Immune inhibitory receptors. Science
2000; 290; 84–89.
8. DeLisser HM, Newman PJ, Albelda SM. Molecular and functional
aspects of PECAM-1 ⁄ CD31. Immunol. Today 1994; 15; 490–495.
9. Falini B, Tiacci E, Pucciarini A et al. Expression of the IRTA1
receptor identifies intraepithelial and subepithelial marginal zone
B cells of the mucosa-associated lymphoid tissue (MALT). Blood
2003; 102; 3684–3692.
10. Isaacson PG, Norton AJ. Mucosa-associated lymphoid tissue
(MALT) and the MALT lymphoma concept. In: Isaacson PG,
12 B Falini et al.
Norton AJ, eds. Extranodal Lymphomas. Edinburgh: Churchill
Livingstone, 1994; 5–14.
11. Sheibani K, Fritz RM, Winberg CD, Burke JS, Rappaport H.
‘Monocytoid’ cells in reactive follicular hyperplasia with and
without multifocal histiocytic reactions: an immunohistochem-
ical study of 21 cases including suspected cases of toxoplasmic
lymphadenitis. Am. J. Clin. Pathol. 1984; 81; 453–458.
12. Stein H, Lennert K, Mason DY, Liangru S, Ziegler A. Immature
sinus histiocytes. Their identification as a novel B-cell population.
Am. J. Pathol. 1984; 117; 44–52.
13. Kojima M, Nakamura S, Itoh H et al. Occurrence of monocytoid
B-cells in reactive lymph node lesions. Pathol. Res. Pract. 1998;
194; 559–565.
14. Falini B, Fizzotti M, Pucciarini A et al. A monoclonal antibody
(MUM1p) detects expression of the MUM1 ⁄ IRF4 protein in a
subset of germinal center B cells, plasma cells, and activated T
cells. Blood 2000; 95; 2084–2092.
15. Flenghi L, Bigerna B, Fizzotti M et al. Monoclonal antibodies PG-
B6a and PG-B6p recognize, respectively, a highly conserved and
a formol-resistant epitope on the human BCL-6 protein amino-
terminal region. Am. J. Pathol. 1996; 148; 1543–1555.
16. Went P, Agostinelli C, Gallamini A et al. Marker expression in
peripheral T-cell lymphoma: a proposed clinical-pathologic
prognostic score. J. Clin. Oncol. 2006; 24; 2472–2479.
17. Pileri SA, Roncador G, Ceccarelli C et al. Antigen retrieval
techniques in immunohistochemistry: comparison of different
methods. J. Pathol. 1997; 183; 116–123.
18. Cordell JL, Falini B, Erber WN et al. Immunoenzymatic labeling of
monoclonal antibodies using immune complexes of alkaline
phosphatase and monoclonal anti-alkaline phosphatase (APAAP
complexes). J. Histochem. Cytochem. 1984; 32; 219–229.
19. Haralambieva E, Banham AH, Bastard C et al. Detection by the
fluorescence in situ hybridization technique of MYC transloca-
tions in paraffin-embedded lymphoma biopsy samples. Br. J.
Haematol. 2003; 121; 49–56.
20. Ventura RA, Martin-Subero JI, Jones M et al. FISH analysis for the
detection of lymphoma-associated chromosomal abnormalities in
routine paraffin-embedded tissue. J. Mol. Diagn. 2006; 8; 141–151.
21. Campo E, Pileri S, Jaffe ES, Muller-Hermelink HK, Nathwani BN.
Nodal marginal zone lymphoma. In Swerdlow SH et al. eds.
World Health Organization classification of tumours of haematopoietic
and lymphoid tissues. Geneva: IARC Press, 2008; 218–219.
22. Rinaldi A, Mian M, Chigrinova E et al. Genome-wide DNA
profiling of marginal zone lymphomas identifies subtype-specific
lesions with an impact on the clinical outcome. Blood 2010; 117;
1595–1604.
23. Isaacson P, Wright DH. Malignant lymphoma of mucosa-
associated lymphoid tissue. A distinctive type of B-cell lym-
phoma. Cancer 1983; 52; 1410–1416.
24. Isaacson PG, Chott A, Nakamura S, Muller-Hermelink HK, Harris
NL, Swerdlow SH. Extranodal marginal zone lymphoma of
mucosa-associated lymphoid tissue (MALT lymphoma). In
Swerdlow SH et al. eds. World Health Organization classification
of tumours of haematopoietic and lymphoid tissues. Geneva: IARC
Press, 2008; 214–217.
25. Garcia M, Konoplev S, Morosan C, Abruzzo LV, Bueso-Ramos CE,
Medeiros LJ. MALT lymphoma involving the kidney: a report of
10 cases and review of the literature. Am. J. Clin. Pathol. 2007;
128; 464–473.
26. Isaacson PG, Chan JK, Tang C, Addis BJ. Low-grade B-cell
lymphoma of mucosa-associated lymphoid tissue arising in the
thymus. A thymic lymphoma mimicking myoepithelial sialade-
nitisf. Am. J. Surg. Pathol. 1990; 14; 342–351.
27. Takagi N, Nakamura S, Yamamoto K et al. Malignant lymphoma
of mucosa-associated lymphoid tissue arising in the thymus of a
patient with Sjogren’s syndrome. A morphologic, phenotypic,
and genotypic study. Cancer 1992; 69; 1347–1355.
28. Mosnier JF, Brousse N, Sevestre C et al. Primary low-grade B-cell
lymphoma of the mucosa-associated lymphoid tissue arising in
the gallbladder. Histopathology 1992; 20; 273–275.
29. van den Oord JJ, de Wolf-Peeters C, Desmet VJ. The marginal
zone in the human reactive lymph node. Am. J. Clin. Pathol.
1986; 86; 475–479.
30. Stein K, Hummel M, Korbjuhn P et al. Monocytoid B cells are
distinct from splenic marginal zone cells and commonly derive
from unmutated naive B cells and less frequently from postger-
minal center B cells by polyclonal transformation. Blood 1999;
94; 2800–2808.
31. Camacho FI, Garcia JF, Sanchez-Verde L et al. Unique pheno-
typic profile of monocytoid B cells: differences in comparison
with the phenotypic profile observed in marginal zone B cells
and so-called monocytoid B cell lymphoma. Am. J. Pathol.
2001; 158; 1363–1369.
32. Sheibani K, Sohn CC, Burke JS, Winberg CD, Wu AM, Rappaport
H. Monocytoid B-cell lymphoma. A novel B-cell neoplasm. Am. J.
Pathol. 1986; 124; 310–318.
33. Dono M, Burgio VL, Tacchetti C et al. Subepithelial B cells in the
human palatine tonsil. I. Morphologic, cytochemical and pheno-
typic characterization. Eur. J. Immunol. 1996; 26; 2035–2042.
34. Dono M, Zupo S, Augliera A et al. Subepithelial B cells in the
human palatine tonsil. II. Functional characterization. Eur. J.
Immunol. 1996; 26; 2043–2049.
35. Dono M, Zupo S, Massara R et al. In vitro stimulation of human
tonsillar subepithelial B cells: requirement for interaction with
activated T cells. Eur. J. Immunol. 2001; 31; 752–756.
36. Lazzi S, Bellan C, Tiacci E et al. IRTA1+ monocytoid B cells in
reactive lymphadenitis show a unique topographic distribution
and immunophenotype and a peculiar usage and mutational
pattern of IgVH genes. J. Pathol. 2006; 209; 56–66.
37. Mebius RE, Nolte MA, Kraal G. Development and function of the
splenic marginal zone. Crit. Rev. Immunol. 2004; 24; 449–464.
38. Spencer J, Perry ME, Dunn-Walters DK. Human marginal-zone B
cells. Immunol. Today 1998; 19; 421–426.
39. Attygalle AD, Liu H, Shirali S et al. Atypical marginal zone
hyperplasia of mucosa-associated lymphoid tissue: a reactive
condition of childhood showing immunoglobulin lambda light-
chain restriction. Blood 2004; 104; 3343–3348.
40. Kojima M, Morita Y, Shimizu K et al. Immunohistological
findings of suppurative granulomas of Yersinia enterocolitica
appendicitis: a report of two cases. Pathol. Res. Pract. 2007; 203;
115–119.
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Figure S1. Colonized B-cell follicle in a nodal mar-
ginal zone lymphoma (MZL).
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supporting materi-
als supplied by the authors. Any queries (other than
missing material) should be directed to the correspond-
ing author for the article.
 2012 Blackwell Publishing Ltd, Histopathology