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LABORATORY INVESTIGATION
Vol. 81, No. 7, p. 961, 2001
Copyright © 2001 by The United States and Canadian Academy of Pathology, Inc.
Printed in U.S.A.
Biclonality of Gastric Lymphomas
Antonello D. Cabras, Sonja Candidus, Falko Fend, Marcus Kremer, Stephan Schulz,
Cesare Bordi, Gregor Weirich, Heinz Höfler, and Martin Werner
Departments of Pathology (SC, FF, MK, SS, GW, HH, MW), Technische Universität München, München, Germany;
and GSF-National Research Center for Environment and Health, Neuherberg (ADC, HH), Germany, and Parma (CB),
Italy
SUMMARY: The pathogenesis and clonal evolution of gastric diffuse large B-cell lymphoma (DLBCL) and its relationship to
extranodal marginal zone B-cell lymphoma (MZBL), mucosa-associated lymphoid tissue (MALT) type, are still controversial. The
aim of this study was to establish the clonality of morphologically distinct areas of gastric lymphomas as well as their genetic
relationship to each other. Six gastric lymphomas, consisting of two MZBL, MALT type, two DLBCL, and two “composite”
lymphomas were subjected to laser capture microdissection and subsequent PCR-based amplification of the immunoglobulin
heavy chain gene. One DLBCL showed a biclonal pattern of rearranged immunoglobulin heavy chain (IgH) genes of two different
areas without evidence of a common origin. Two composite DLBCL with areas of extranodal MZBL, MALT type, were also
biclonal and displayed different IgH gene rearrangements in the small-cell and in the large-cell components, respectively.
Sequencing of the CDR3 region revealed unique VH-N-D and D-N-JH junctions, thus corroborating the presence of two genuinely
distinct tumor clones in each of these three cases. In contrast, the remaining three gastric lymphomas (one DLBCL and two
MZBL, MALT type) showed IgH gene rearrangements in which CDR3 regions were identical in the different tumor areas. Our
results suggest that gastric DLBCL may be composed of more than one tumor cell clone. Further, DLBCL may not necessarily
evolve by transformation of a low-grade lymphoma, but may also originate de novo. An ongoing emergence of new tumor clones
may considerably hamper molecular diagnosis and follow-up of gastric DLBCL. (Lab Invest 2001, 81:961–967).
he two main groups of gastric lymphoma are
extranodal marginal zone B-cell lymphoma
MZBL), mucosa-associated lymphoid tissue (MALT)
type, and diffuse large B-cell lymphoma (DLBCL)
Jaffe et al, 1999). MZBL, MALT type, is composed of
a monotonous, diffuse infiltrate of small lymphoid
cells. DLBCL is characterized by a diffuse infiltrate of
large malignant lymphoid blasts resembling centro-
blasts, plasmablasts, and immunoblasts. Occasion-
ally, DLBCL shows both small- and large-cell compo-
nents. These tumors have been called “composite”
lymphomas, their histologic spectrum ranging from
cases with well separated small- and large-cell com-
ponents, to tumors in which these components are
intermingled (De Jong et al, 1997; De Wolf-Peeters
and Achten, 1999).
small-cell areas in the same gastric B-cell lymphoma
have demonstrated a common clonal origin of both
components (Chan et al, 1990; Du et al, 1996a, 1996b;
Peng et al, 1997). These results suggest that the
DLBCL may have developed by transformation of the
MZBL, MALT type, component (De Wolf-Peeters and
Tierens, 1998). In contrast, Matolcsy et al (1999) found
no clonal relationship between well separated small-
cell and large-cell components of one case of a
multifocal gastric lymphoma.
The aim of the present study was to establish the
clonality in different areas of two gastric MZBL, MALT
type, two gastric DLBCL, and two composite gastric
DLBCL with areas of MZBL, MALT type, as well as
their genetic relationships with one another.
T
(
(
The histogenesis of gastric DLBC lymphoma, as
well as its possible clonal relationship to concomitant
or preceding low-grade components, has been
strongly debated recently and is still not fully clarified.
The clonality of B-cell non-Hodgkin’s lymphomas can
be assessed by the PCR analysis of rearranged im-
munoglobulin heavy chain (IgH) genes that vary in size
in different neoplastic cell populations (Trainor et al,
Results
PCR, Agarose Gel Analysis
PCR amplification of the IgH genes in the different
microdissected tumor samples yielded sharp bands of
the same sizes in the two MZBL, MALT type (Cases 1
and 2), as well as in the two DLBCL (Cases 3 and 4).
PCR amplification yielded bands of varying sizes in the
two DLBCL with areas of MZBL, MALT type only
1991). Several studies comparing the patterns of
clonal IgH gene rearrangement between large-cell and
(Cases 5 and 6). A representative example is shown in
Figure 1 (Case 5). The dominant clones of each tumor
area were found in PCR of both the microdissected
tissues and the entire sections. In Cases 5 and 6,
however, PCR-based DNA amplification of the entire
sections only revealed the clone of the large-cell
component. PCR amplification of DNA derived from
Received January 29, 2001.
Address reprint requests to: Dr. Antonello D. Cabras, Institute of Pathology,
GSF-National Research Center for Environment and Health, Ingolstädter Land-
strasse 1, D-85764 Neuherberg, Germany. E-mail: ad.cabras@lrz.tum.de
Laboratory Investigation • July 2001 • Volume 81 • Number 7 961

Cabras et al
ment (Case 4, Fig. 2B). Also, small- and large-cell
components of the two composite lymphomas
showed individual sequences of VH-(N)-D and
D-(N)-JH junctions (Cases 5 and 6, Fig. 2B).
Intraclonal Variations
In one composite lymphoma (Case 6), multiple se-
quence variations were identified among different
PCR clones derived from the small-cell component:
two replacement (R) and two silent (S) mutations were
observed in the D-region. One R- and three
S-mutations, and a deletion of three nucleotides were
observed in the joining region (Fig. 2B).
Discussion
Gastric DLBCL are thought to develop either as a
clonal progression from a preexisting MZBL, MALT
type, tumor or independently from the coexisting
small-cell components, ie de novo (De Wolf-Peeters
and Achten, 1999; Isaacson 1996). Evidence for both
hypotheses has been drawn from the detection of
either identical (Chan et al, 1990; Du et al 1996a,
Figure 1.
Ethidium bromide–stained 3% agarose gel of Frame Region 3, PCR products
from Case 5. Lane MW, molecular-weight marker; Lane 1, diffuse large B-cell
lymphoma (DLBCL) component; Lane 2, marginal zone B-cell lymphoma
(
MZBL) component; Lane 3, polyclonal control; Lane 4, monoclonal control.
reactive hyperplastic lymphoid tissue (chronic tonsilli-
tis) revealed a polyclonal pattern with and without
microdissection (data not shown). PCR amplicons
ranged from 83 to 128 bp. PCR products from differ-
ent tumor areas were identical in size (92 bp in Case 1,
1996b) or distinct (Matolcsy et al, 1999) B-cell clones
in composite DLBCL with coexisting MZBL, MALT
type. Whereas these previous studies aimed at the
genetic relationship between small- and large-cell
components in relatively rare composite lymphomas,
the question of whether gastric DLBCL without a
concomitant small-cell component are monoclonal or
oligoclonal malignancies has not yet been addressed.
In our study, three gastric lymphomas (two compos-
ite lymphomas and one DLBCL) exhibited biclonal IgH
genes. The fact that both unique sequences in each of
these tumors were in frame indicates the presence of
two different tumor clones in each lymphoma. Based
on the isolation of pure tumor cell populations by laser
capture microdissection (LCM), these sequences were
truly derived from tumor cells alone. One composite
lymphoma (Case 5) with well-separated large-cell and
small-cell areas, exhibited distinct CDR3 regions in
both, indicating that neither cell type had evolved from
the other. Immunohistochemical demonstration of Ig
light chain restriction in the small-cell but not the
large-cell component further corroborated the genetic
findings. A similar situation was found in the second
composite lymphoma (Case 6), in which, in contrast to
the above-mentioned case, large and small tumor
cells were intermingled. After being separated by
LCM, these cell types also displayed distinct CDR3
regions. Therefore, the DLBCL components in these
two composite lymphomas seem not to have arisen
from transformed cells of the adjacent MZBL, MALT
type, but rather independently. It is therefore not
unlikely that DLBCL in composite lymphomas origi-
nates de novo rather than evolving by transformation
of a low-grade lymphoma. However, theoretically,
these DLBCL components may also have arisen from
transformed low-grade progenitors no longer present
in the tumor. Histologically, one of the three biclonal
tumors, a DLBCL, showed phenotypically identical
128 bp in Case 2, 98 bp in Cases 3 and 4, respectively)
in the two MZBL, MALT type (Cases 1 and 2), and the
two DLBCL (Cases 3 and 4). PCR products from
well-separated components differed in size in one
composite lymphoma (Case 5; 119 bp in the DLBCL
and 95 bp in the MZBL, MALT type). PCR products of
distinct sizes were also identified in a second com-
posite lymphoma with intermingling MZBL, MALT
type, and DLBCL components (95 bp in the MZBL,
MALT type, component and 83 bp in the DLBCL
component).
Sequence Analysis
All CDR3 sequences were in frame. The nucleotide
and deduced amino acid sequences are shown in
Figure 2, A and B. D- and JH-genes were detected in
all six cases (11 specimens). Four D-genes matched
database sequences (germline), whereas another
seven showed single nucleotide deviations from data-
base entries (20% and less). Fusions of two D-regions
were found in two cases (3 and 5). No preferential
utilization of D-genes was apparent, whereas JH4
segments were identified in five cases (seven speci-
mens). In six specimens, germline JH genes were
found, whereas sequence deviations ranging from
75% to 96% were apparent in three specimens.
In both MZBL, MALT type, as well as in one DLBCL,
identical sequences of the D- and JH segments were
found in specimens from antrum and corpus mucosa
(
Cases 1, 2, and 3, Fig. 2A). In the second DLBCL,
however, tumor infiltrates of antrum and corpus mu-
cosa displayed different sequences of VH-(N)-D and
D-(N)-JH junctions, solely present in each compart-
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62 Laboratory Investigation • July 2001 • Volume 81 • Number 7

Biclonality of Gastric Lymphomas
Figure 2.
A, Genetically identical lymphoma infiltrates from different locations: tumor-specific sequences of CDR3 gene segments and translated peptides in two monoclonal
MZBL, mucosa-associated lymphoid tissue (MALT) type (Cases 1 and 2), and in one monoclonal DLBCL (Case 3). The sequences are grouped and subdivided into
VH, N, D, N, and JH regions. The names of the germline D and JH genes with maximum homology to the segments used in the VDJ joining are shown in parentheses
on the appropriate position. B, Genetically heterogenous lymphoma infiltrates: tumor-specific sequences of CDR3 gene segments and translated peptides in one
biclonal DLBCL (Case 4), and in two biclonal “composite” DLBCL (Cases 5 and 6). The sequences are grouped and subdivided into VH, N, D, N, and JH regions. The
names of the germline D and JH genes with maximum homology to the segments used in the VDJ joining are shown in parentheses on the appropriate position.
Dashes (Case 6): identity between the nucleotides of the IgH gene sequences. The intraclonal variations are shown by using uppercase letters for replacement
mutations and lowercase letters for silent mutations. In Cases 1 to 5, the sequence variations are underlined.
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Cabras et al
lymphoid blasts at all sites. In this case, the microdis-
sected tumor cells from antrum and corpus mucosa
showed different VH-N-D and D-N-JH junctions as
evidence of distinct tumor clones.
context of Helicobacter pylori (HP) infection, which
was detected in these cases. On the other hand, the
three monoclonal gastric lymphomas were negative
for HP. These findings support an HP-linked patho-
genesis of gastric lymphomas (Wotherspoon et al,
1991). Further evidence of this pathway stems from
the finding of single nucleotide variations in the CDR3
region of one composite lymphoma (Case 6). Similar
variations have previously been interpreted as indica-
tors of a postgerminal center origin (Du et al 1996a,
1996b; Hallas et al, 1998). An autoimmune response,
chiefly triggered by HP-associated immune phenom-
enoma, may also play an important role in the devel-
opment of gastric lymphomas (Greiner et al, 1994;
Hussell et al, 1996). Chronic HP-gastritis may lead to
non-neoplastic B-cell proliferation in the mucosa (Na-
kamura et al, 1998), the neoplastic transformation of
which is dependent on mutations in tumor suppressor
genes or oncogenes (Neumeister et al 1997; Peng et al
1996). Ott et al (1994) showed that inflammatory
lymphoid infiltrates outside the tumor mass displayed
additional clonal B-cell populations unrelated to the
primary tumor in two cases of gastric MZBL, MALT
type. These authors hypothesized that additional
clonal cell populations may develop apart from the
primary tumor caused by continued triggering by the
autoimmune response. Our biclonal gastric lympho-
mas may have developed from different non-
neoplastic clonal cell populations in HP-positive gas-
tritis leading to unrelated tumor clones.
Biclonality in different cell compartments of the
same tumor or at different anatomical sites may be
evidence of ongoing antigenic stimulation. In all tumor
samples except for both composite lymphomas, we
were able to detect the same clonal products from
entire sections as well as from the microdissected
tissues. These results suggest that the amplificates
from the microdissected samples were not derived
from minor clonal populations of uncertain biologic
significance, or PCR artifacts, but in fact represent the
malignant cells. In the two composite lymphomas,
only one of the two clones obtained from microdis-
sected tissue was detectable in PCR from whole
sections. This fact probably reflects preferential am-
plification, either due to different amplification efficien-
cies based on differences in the primer binding sites or
to different sizes of the malignant clones.
In our study, DNA was obtained by LCM, a tech-
nique that was not used in previous studies on gastric
lymphomas. This may explain why most previous
studies found evidence for the presence of only a
single tumor cell population in gastric lymphomas
(
1
Aiello et al, 1999; Nakamura et al, 1998; Peng et al,
997). Also, interpretation of PCR products of differing
sizes as being polyclonal (Torlakovic et al, 1997) may
explain why oligoclonality has not been detected more
frequently in gastric lymphomas.
Biclonality of gastric DLBCL may hamper the mo-
lecular genetic analysis of these tumors. PCR analysis
of the IgH gene for the detection of minimal residual
disease or relapse after therapy may reveal clones
differing from those found in the primary tumor. Fi-
nally, in biclonal tumor cell populations, the molecular
biological tumor characteristics, such as specific gene
mutations or the pattern of gene expression associ-
ated with tumor prognosis or therapeutic response,
may be rather heterogeneous.
Biclonality of non-Hodgkin’s lymphomas with unre-
lated tumor clones arising at the same anatomical site
has already been demonstrated in a few nodal and
extranodal lymphomas by PCR IgH gene analyses.
Biclonality of the rare nodal or extranodal composite
lymphomas has been reported previously and it was
suggested that different clones within such tumors
originate from different progenitor cells (Fend et al,
1999). DLBCL arising in the setting of a concomitant
small-cell lymphoma has also occasionally been found
as distinct clonal tumor cell populations in both nodal
and extranodal sites (Matolcsy et al, 1995, 1999).
Biclonality in phenotypically identical tumor areas at
the same anatomic site as found in one of our gastric
DLBCL, however, has only rarely been demonstrated
before. Sklar et al (1984) detected biclonal follicular,
small cleaved-cell lymphomas in single lymph nodes
from two patients. In another study, one case of a
gastric MZBL, MALT type, was found to harbor two
tumor clones with differently rearranged CDR3 regions
Materials and Methods
Material and Clinical Data
Ten formalin-fixed, paraffin-embedded tissue blocks
from six gastric lymphomas were retrieved from the
files of the Department of Pathology, University of
Parma. The material was derived from gastrectomy
specimens. The histologic classification of lympho-
mas was carried out in accordance with defined
histopathologic criteria (Jaffe et al, 1999) using 5-␮m
sections stained with hematoxylin and eosin and Gi-
emsa. Specimens consisted of two MZBL, MALT type,
two DLBCL, and two composite DLBCL with areas of
MZBL, MALT type, lymphoma. Of the two composite
lymphomas, one case (Case 5) had well separated
small-cell and large-cell components, whereas in the
other case (Case 6) these cell types were intermingled.
Tumor blocks from antrum and corpus mucosa were
analyzed in Cases 1 to 4, and from antrum mucosa in
Cases 5 and 6. The tumor cells of the six cases
(
Zucca et al, 1998). Another case of MZBL, MALT
type, of the salivary gland showed genuinely different
clones with distinct CDR3 regions and VH and DH
genes in consecutive biopsy specimens (Lasota and
Miettinen, 1997). The coexistence of phenotypically
identical tumor clones with different rearranged IgH
genes in low-grade B-cell tumors are in accordance
with our findings of more than one neoplastic tumor
clone in gastric DLBCL.
The presence of biclonality in three gastric lympho-
mas (Cases 4 to 6) is especially interesting in the
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64 Laboratory Investigation • July 2001 • Volume 81 • Number 7

Biclonality of Gastric Lymphomas
examined were immunohistochemically positive for
CD20 and CD79a, and negative for CD3, CD5, CD10,
CD23, IgA, IgG, IgD, IgM, and ␭-chain. Light chain
restriction (␬) was found in one DLBC lymphoma (Case
with the lymphoma samples. As a polyclonal control,
DNA isolated from paraffin sections of a case of
chronic tonsillitis was used. For all tumor and control
samples, the PCR analyses from the DNA samples
obtained with and without tissue microdissection were
performed in three independent experiments.
4) and in the small-cell component (␬) of a composite
lymphoma (Case 5). Three cases were HP-positive by
immunohistochemistry. The Musshoff modification of
the Ann Arbor system was used for tumor staging
Agarose Gel Electrophoresis
(
Musshoff, 1977). Table 1 summarizes the histopatho-
logic and clinical data.
The PCR amplificates were electrophoresed on 2.5%
agarose gels (FMC BioProducts, Rockland, Maine) for
the ␤-actin pseudogene, and on 3% intermediate-
melting agarose gels (Metaphor; FMC BioProducts)
for the IgH gene rearrangements, followed by DNA
visualization with ethidium bromide.
Preparation of DNA
3
The tumor cells (3 ϫ 10 per sample) were isolated
under microscopic control by LCM from 10 ␮m tissue
sections previously stained with hematoxylin and eo-
sin for 15 seconds. LCM was performed using a
PixCell laser capture microscope (Arcturus Engineer-
ing, Santa Clara, California) as described previously
Sequencing of the CDR3 Region
For purification, PCR-products were electrophoresed
on low-melting agarose gels (NuSieve GTG agarose
1.5%; FMC BioProducts). Twelve microdissected
samples from six patients yielded sharp clonal bands
that were excised and amplicons recovered using
QIAquick Gel extraction kit (Qiagen GmbH, Hilden,
Germany). The purified PCR products were further
processed with Pfu DNA polymerase for blunt ends,
ligated into pPCR-Script amp SK(ϩ) cloning vector,
and then used for transformation of E. coli according
to the manufacturer’s protocol (XL10-Gold Kan ultra-
competent cells, PCR-Script amp cloning kit; Strat-
agene Cloning Systems, La Jolla, California). For each
PCR fragment, 10 clones were selected and se-
quenced. The plasmids were purified using QIAprep
Miniprep Kit (Qiagen) and cycle sequenced using a
plasmid specific forward primer (T3; Stratagene). Cy-
cle sequencing was performed using BIG dye-deoxy
terminators (Applied Biosystems, Foster City, Califor-
nia). Sequencing reactions were separated on 10%
polyacrylamide gels (Long Ranger; FMC BioProducts)
in an automated sequencer (ABI 377; Applied
Biosystems).
(
Fend et al, 1999) (Fig. 3). In the DLBCL and MZBL,
MALT type, the tumor cells were isolated from two
different tumor blocks, ie corpus and antrum (two
samples per case), and in the composite lymphomas
from DLBCL and MZBL, MALT type, components of
one tumor block (two samples per case), respectively.
To investigate whether the dominant clone(s) found in
the microdissected samples were also detectable in
whole tissue sections, DNA was isolated from consec-
utive sections of all 10 tissue blocks without microdis-
section. DNA-quality was assessed by amplification of
the ␤-actin pseudogene (Ghossein et al, 1994; Moos
and Gallwitz, 1983). For all PCR experiments, a metal
block thermal cycler was used (PTC-200; MJ Re-
search, Watertown, Massachusetts).
IgH Gene Rearrangements
DNA amplification for the IgH gene rearrangements
was carried out as a single-step PCR according to a
published protocol (Segal et al, 1994). Primer se-
quences were FR3: 5'- ACA CGG C(C/T)(G/C) TGT
ATT ACT GT-3' and LJH: 5'-TGA GGA GAC GGT GAC
C-3'. A monoclonal positive control (isolated from
paraffin sections of a case of nodal DLBCL) and a
The sequences of the expressed D- and JH-
segments were compared with those of the published
germline D-, DIR-, and JH-segments using the DNA-
SIS v.2.6 program and the IMGT database (Lefranc
negative control (H O instead of DNA) were amplified
2
Table 1. Clinicopathological Data of Cases Analyzed
Classification
MZBL
Case 1
Case 2
Staging
M/F
Age
HP
Follow Up
EI1
EII1
F
M
50
70
neg
neg
27 months AWD
7 months ANED
DLBCL
Case 3
Case 4
EII1
EI2
M
M
66
72
neg
pos
156 months ANED
8 months DOD
Composite
DLBCL/MZBL
Case 5
EI2
EI2
M
M
61
59
pos
pos
120 months ANED
142 months ANED
Case 6
M/F, male/female; HP, Helicobacter pylori; DOD, dead of disease; ANED, alive with no evidence of disease; AWD, alive with disease; MZBL, marginal zone B-cell
lymphoma; DLBCL, diffuse large B-cell lymphoma.
Laboratory Investigation • July 2001 • Volume 81 • Number 7 965

Cabras et al
Figure 3.
A, MZBL component of Case 5 before laser capture microdissection (LCM). B, MZBL component of Case 5 after LCM. C, DLBCL component of Case 5 before LCM.
D, DLBCL component of Case 5 after LCM.
et al, 1999). The sequences were aligned using the
stringent criteria defined by Corbett et al (1997).
De Jong D, Boot H, Van Heerde P, Hart GAM, and Taal BG
1997). Histological grading in gastric lymphoma: Pretreat-
ment criteria and clinical relevance. Gastroenterology 112:
466–1474.
(
1
Acknowledgement
De Wolf-Peeters C and Achten R (1999). The histogenesis of
large-cell gastric lymphomas. Histopathology 34:71–75.
The authors are especially thankful to Birgit Geist for
expert technical assistance.
De Wolf-Peeters C and Tierens A (1998). Controversies in
MALT lymphoma classification, low and high grade. Histo-
pathology 32:277–278.
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