Pathology International 1999; 49: 752–758
Case Report
Diffuse large B cell lymphoma expressing the natural killer cell
marker CD56
Toru Sekita,1 Jun-ichi Tamaru,1,3 Kouichi Isobe,1 Kenichi Harigaya,1 Syu-ichi Masuoka,2
Toshio Katayama,2 Masayuki Kobayashi2 and Atsuo Mikata1,3
1
First Department of Pathology, School of Medicine, Chiba University, Chiba, 2Department of General Internal Medicine,
Kashiwa Hospital, Jikei University, School of Medicine, Kashiwa and 3Department of Pathology, Saitama Medical
Center, Saitama Medical School, Kawagoe, Japan
The expression of the natural killer (NK) cell antigen, CD56,
in hematological malignancies is rare. However, there are
several reports that some hematological malignancies,
such as T/NK cell lymphoma, multiple myeloma (MM) and
acute myeloid leukemia (AML), express this molecule. In B
cell non-Hodgkin’s lymphomas (NHL), however, very limited
number of cases have been reported to express CD56
molecule. Although one study has recently described that
half of microvillous B cell lymphoma (MVL), an uncommon
subset of large cell lymphoma, expressed CD56, there have
been no reports about most common type of B-NHL, diffuse
large B cell lymphoma (DLBL) other than a mention of weak
CD56 expression in one of 83 DLBL. We herein presented
the first case of diffuse large B cell lymphoma expressing
CD56 clearly. The immunophenotype determined by
immunostaining and flow cytometric analysis was CD101,
CD191, CD201, CD45RO2, CD32 and CD561. On immuno-
histochemical study, neither bcl-2 nor TIA-1 was positive for
tumor cell. Monoclonal immunoglobulin heavy chain (IgH)
gene rearrangement was detected, and the sequence
analysis of the variable region of IgH (VH) suggested that
this tumor was derived from antigen selected post germinal
center B cell. Conventional combination chemotherapy
(CHOP) was administered, and the patient has still been in
complete remission for 10 months.
Key words: CD56, diffuse large B cell lymphoma, immunoglob-
ulin heavy chain gene rearrangement, variable region of im-
munoglobulin heavy sequence
The nerve cell adhesion molecule (NCAM), CD56, is a
member of the immunoglobulin superfamily, and can be
Correspondence: Dr Toru Sekita, First Department of Pathology,
School of Medicine, Chiba University, Internal Medicine, Kanagawa
Rehabilitation Center, 516 Nanasawa, Atugi City, Kanagawa 243-
0121, Japan.
Received 21 January 1999. Accepted for publication 20 March
1999.
detected on neuron, astrocyte, Schwann cell, myotube and
natural killer (NK) cell.1–4 The NCAM is a glycoprotein con-
taining a single polypeptide chain and plays an important role
in a variety of morphogenetic events in the nervous system.
In addition, the NCAM also appears to be involved in signal
transduction pathways.5–7 The NCAM has been detected on a
number of neoplasms (e.g. neuroectodermal tumors, NK/T
cell lymphoma/leukemia, multiple myeloma (MM), and acute
myeloid leukemia (AML)).8–20 In these literatures, the
researchers have described that the CD56 expression was
associated with frequent extranodal involvement in NK-cell
lymphoma,13,14 clinical behavior in MM19,20 and prognosis in
AML.17
In B cell non-Hodgkin’s lymphoma (NHL), there have been
reported only six cases expressing CD56 on immunostaining.
A case of B-lymphoblastic lymphoma reported by Ng et al.,21
one case of diffuse large B cell lymphoma (DLBL) reported by
Kern et al.,13 and four cases by Hammer et al.22 Hammer et
al. have recently described that four cases out of eight
microvillous lymphoma (MVL), an uncommon subset of large
cell lymphoma, expressed CD56.22 Although the details of the
other two cases were not described in their literatures,
Hammer et al. have analyzed in detail and suggested that the
expression of CD56 was involved in a sinusal growth pattern
seen in MVL.22 However, the biological role and the mech-
anism of the CD56 expression in these B cell lymphomas still
remain unknown due to its rarity. Therefore, it is very
important to know how this molecule is expressed in B-NHL,
and whether these CD56-expressing tumors possess char-
acteristic features similar to those observed in NK/T cell
lymphoma or MVL. Also, it seems to be important to
recognize that DLBL, one of the most popular subtype of
NHL, express CD56.
In the present study, we present a 16-year-old patient with
CD56-positive diffuse large B cell lymphoma, and investigate
histologic feature, surface marker and karyotype for eluci-
 CD56-positive DLBL 753

dating biological role and mechanism of CD56 expression. Cytogenetic analysis

Furthermore, we analyze the rearranged immunoglobulin
heavy (IgH) chain gene variable region (VH) to determine the
tumor cell derivation.
CASE REPORT
Cytogenetic study was carried out after 72 h fluorodeoxy
uridine synchronized culture of the lymph node cells in the
presence of 0.2 mL/10 mL phytohemagglutinin. Metaphase
chromosomes were banded using the trypsin-Giemsa
method.

A 16-year-old male visited our hospital with a complaint of

Genomic DNA preparation
right cervical lymphadenopathy in August 1997. He was
diagnosed as having DLBL at biopsy from an enlarged right
The DNA was extracted from 10 µm thick paraffin section,
cervical lymph node. An enlarged right cervical node (2.5 cm
by using DEXPAT TM (Takara Biomedicals, Shiga, Japan),
3 3.5 cm) was the only finding at initial presentation, and he
following the manufacturer’s instructions.
was afebrile. The laboratory examination and bone marrow
biopsy did not show any abnormalities. Imaging studies
including standard posteroanterior and lateral chest X-
Polymerase chain reaction amplification
ray, abdominal X-ray, whole body computed tomography,
abdominal ultrasonography and gallium scintigraphy showed
A nested polymerase chain reaction (PCR) was performed
no evidence of metastasis or invasion of the disease. He was
with primers of a mixture of family-specific framework region
diagnosed as stage I DLBL, and was administered six
FR123 and JH primer LJH24 in the first round of amplification,
courses of Conventional combination chemotherapy (CHOP) and consensus primers FR2A and VLJH for reamplification.24
chemotherapy. His course has been favorable to date, and a Polymerase chain reaction conditions were the same as
complete remission has been maintained. previously described.25,26 Eight microliters of PCR product
was subjected to 6% polyacrylamide gel electrophoresis (6%
PAGE) and visualized with ethidium bromide.
MATERIALS AND METHODS

Cloning and sequencing of polymerase chain

Immunophenotypic diagnosis
reaction products
Immunostaining was performed by using streptavidin-
Polymerase chain reaction products were subcloned in the
biotin complex method with 3-39-diamino-benzidine-tetrahy-
pCR 2.1 vector with the original TA Cloning Kit (Invitrogen,
drochloride (DAB) visualization on sections embedded in
San Diego, CA, USA), as the manufacturer’s instructions.
paraffin. Antibodies used for characterization of tumor cells
Plasmid DNA was isolated after overnight culture of randomly
were CD20 (L26), CD45 (LCA), CD45RO (UCHL-1), CD3,
selected colonies using Ultra Clean Mini Plasmid Prep Kit
IgD, IgM and Bcl-2 protein (all from DAKO Japan Ltd, Kyoto,
(MO BIO., Solana Beach, CA, USA). Dye terminator cycle
Japan), CD10 and CD56 (from Novocastra Laboratories Ltd,
sequencing was performed with Ready Reaction Kit (Perkin
Newcastle-upon-Tyne,UK), and TIA-1 (Coulter Immunology,
Elmer, Foster, CA, USA) following the manufacturer’s instruc-
Hialeah, FL, USA).
tions. Nine clones were sequenced from both directions
using the M13 forward and T7 reverse primers. These results
were compared with published sequences of VH germ lines
Flow cytometric assay (Gen Bank, Life Science Tsukuba Research Center, release
104).
Three-color analysis on cell suspensions from the nodal
tissue was performed with the FACScan flow cytometer
(Becton-Dickinson, San Jose, CA, USA) using a gating RESULTS
method by dull-CD45 fluorescence and SCC. Monoclonal
antibodies used were CD3, CD4, CD5, CD8, CD10, CD16, Morphology
CD19 and CD56 (all from Beckman Coulter, Hialeah, FL,
USA), CD56, kappa-chain and lambda-chain (Becton- The normal architecture of the lymph node was completely
Dickinson, San Jose, CA, USA), CD20 (DAKO Japan, Kyoto, effaced and was replaced by large, pleomorphic neoplastic
Japan), and CD34 (Immunotech, Marseille Cedex, France). lymphoid cells. Partially, there were interspersed apoptotic
754 T. Sekita et al.
bodies and tingible-body macrophages in their vicinity. The
neoplastic lymphoid cells had nuclear contour irregularities
with variably clumped chromatin and conspicuous nucleoli
(Fig. 1).
Figure 1 Lymph node biopsy showing effacement of normal
architecture by large atypical lymphoid cells.
Figure 2 Immunohistochemistry of lymph node. (a) Neoplastic cells stain positive for CD56 and (b) CD10.
Immunophenotype analysis
Immunohistochemical studies revealed that the immuno-
phenotypes of the atypical large neoplastic cells were
CD451, CD101, CD201, CD561, CD3-, CD45RO-, IgM-,
IgD-, bcl-2-, and TIA-1 (Fig. 2). The results of the flow cyto-
metric analysis are summarized in Table 1. The majority of
tumor cells were reactive for the B cell marker (CD10, CD19,
CD20, lambda) and CD56 antibodies (Fig. 3). Epstein-Barr
virus was not detected by in situ hybridization (data not
shown).
Cytogenetic analysis
Tumor cells showed various chromosomal abnormalities
(Fig. 6). The partial deletion of 6q, and the addition of 11q
were detected.
PCR amplification and sequence analysis
The PCR product of the rearranged IgH gene showed a
monoclonal band on 6% PAGE (Fig. 4). The result of VH
 CD56-positive DLBL 755

Figure 3 Flow cytometric analysis of double-labeled suspended lymph node cells. The dot plots reveal (a) CD561/CD161 and (b)
CD101/CD341 population of lymph node cells.

Table 1 Percentage of positive lymph node cells

by flow cytometric analysis

Antibody (%)
CD3 20.4
CD4 6.7
CD5 20.2
CD8 11.6
CD10 75.5
CD16 0.2
CD19 81.0
CD20 83.9
CD34 0.4
CD45 70.1
CD56 84.8
K-chain 0.4
L-chain 78.6

Figure 4 Products of immunoglobulin heavy polymerase chain

reaction analyzed on polyacrylamide gel electrophoresis (6%)
sequence is shown in Fig. 5. This case displayed 92% stained with ethidium bromide. M, molecular marker; 1, positive
homology to the germline gene sequence of the VH4 family. control (Raji DNA); 2, negative control (without template DNA); 3,
CD56-positive diffuse large B cell lymphoma.
The replacement and silent mutations in the VH segments
are summarized in Table 2. The replacement/silent mutation
Table 2 Distribution of mutations in variable region gene
(R/S) ratio of the complementarity-determining region (CDR)
was 4.0 (. 2.9) and the framework region (FW) was 0.4 CDR2 FW3 Total (%) R/S ratio
R S R S R S CDR2 FW3
(, 1.5). Of the nine clones sequenced, three contained
single mutation in each with a mutation rate of approximately 4 1 2 5 6 (4.2%) 6 (4.2%) 4 0.4
0.2%. Germline usage: V4.18; homology: 91.6%; R/S ratio, replacement/
silent mutation ratio; FW, framework region.

DISCUSSION only two reports that mentioned the expression of CD56

immunohistochemically in B-NHL, a B-lymphoblastic lym-
We have reported the first case of a DLBL, whose tumor cells phoma reported by Ng et al.21 and DLBL reported by Kern et
clearly expressed surface CD56. Except for the cases of al.13 However, the details of these two cases have not been
MVL, which is a rare type of large cell lymphoma, there were described in their literatures. Recently, Hammer et al.
756 T. Sekita et al.

Figure 5 Sequence analysis of the polymerase chain reaction products variable region (VH) sequence obtained from this case. The first line
shows the amino acid sequence of germline gene. The second line shows the published VH germline gene most homologous to our sequencing
data. The third and following lines show our sequencing data. Replacement mutations are in upper case, while silent mutations are in lower
case.

Figure 6 Trypsin-Giemsa-banded karyotype

of lymphoma cells from this case showing
46,XY,del(6) (q?), del(10)(p11), add(11)
(q2?5)32.

reported in detail that four cases out of eight MVL expressed
CD56.22 They have suggested that CD56 expression in MVL
was associated with a cohesive sinusal growth pattern seen
in MVL. The histologic features of MVL have been reported to
show a cohesive sinusal growth pattern of large transformed
cells similar to anaplastic large cell lymphoma, and display
large areas of necrosis in some cases. In electron micro-
scopic examinations, MVL shows predominantly large
transformed lymphocytes with thin villous projections on their
surfaces. The typical histologic findings in our case were
some apoptotic bodies and an increase in tangible body
macrophages in their vicinity. Although we were not able to
assess the presence of villous projection ultrastructurally, our
case was distinct from MVL microscopically. In addition, our
case did not show the evidence of angiocentricity, angio-
destruction, and zonal tumor cell death (interpreted as
‘coagulative necrosis’), which were characteristic findings
seen in NK-cell lymphoma.
From the previous ultrastructural studies, the cell origin of
MVL was presumed to be the transformed follicular center
cell.27 In addition, Hammer et al. demonstrated the presence
of Bcl-2 protein expression in 88% of their cases and
membrane expression of CDw75 in 63%, and suggested a
follicular center origin of MVL. Furthermore, they identified
bcl-2 gene rearrangement in one of three cases examined.
Although our case was CD101 by immunostaining and flow
cytometric analysis, it was negative for bcl-2 protein immuno-
histochemically. In addition, we could not identify t14;18 on
karyotypic analysis. Furthermore, VH region sequence
analysis showed a low to intermediate somatic mutation
without clear evidence of ongoing somatic mutation, although
three clones each contained one point mutation. The R/S
ratio of CDR2 and FW3 was 4, and 0.4, respectively. The
discrepancy of these R/S ratios indicated that tumor cell was
associated with antigen stimulation.28 These VH sequence
analyses suggested that cell origin of our case was a B cell at
a stage after antigen selection, such as memory B cells. From
the standpoint of surface marker and VH sequence analysis,
it is presumed that our case has different derivation from
MVL.
The mechanism of CD56 expression in B-NHL remains
to be understood. CD56 is known to be expressed in NK
cell lineage with a variety of degrees. Furthermore, some
researchers have reported the expression of CD56 in
myeloid leukemia (e.g. in mixed lineage leukemia,16 and t8;21
leukemia).17 They suggested that these tumors may originate
in progenitors at an immature stage that is common to both
the NK cell and the myeloid lineage. However, there have not
been any reports suggesting the presence of a common
progenitor of the B cell and the NK cell lineage. According to
the cytogenetic study, various chromosomal abnormalities
were detected in our case. Of these, we could identify the 11q
aberration. Although we were not able to assess this 11q
CD56-positive DLBL 757
aberration thoroughly, it may be involved in the CD56
expression in this case, because the single NCAM gene is
known to be located on chromosome 11q23. Furthermore, we
could observe 6q deletion in our case. The 6q deletion is
observed in about 20% of NHL.29,30 In particular, two distinct
regions of minimal deletion (RMD), at 6q25–27 (RMD-1)
and at 6q21–23 (RMD-2), have been identified, suggesting
the presence of two tumor suppressor genes.31 However,
the exact break point of the 6q deletion could not be identified
in this case, as it may have been associated with tumori-
genesis.
The biologic significance of CD56 expression in B cell
malignancies is unknown. In NK cell lymphoma, CD56
expression has been reported to be associated with its
extensive extranodal disease.13,14 Baer et al. reported that
AML with CD56 had poor prognosis.17 The role of CD56 in B
cell malignancies is being well clarified in MM. CD56 is
positive in 80–100% of MM and tends to be overex-
pressed,8,12,32,33 whereas Pellat-Deceunynck et al. reported
that dramatic down-expression of CD56 was observed in
advanced extramedullary lesions or in cases that progress to
leukemia.19 These observations suggest that expression of
CD56 and quantitative changes in its expression may be
critical in defining the mechanisms by which plasma cells
adhere or detach from the bone marrow micro-environment.
Hammer et al. suggested that CD56 expression in MVL was
associated with a cohesive sinusal growth pattern.22
However, the biologic role of CD56 expression in our own
case was unclear. An explanation of the significance of CD56
expression in B cell lymphoma should await the accumulation
of cases in the future.
In summary, we have reported the first case of CD56
expressing DLBL. The cell origin of this tumor was assumed
to be antigen-selected post-germinal-center B cells. Although
the biologic role and mechanism of CD56 expression has not
been well understood, the chromosomal abnormalities, 11q
aberration, might be associated with its expression in this
case.
ACKNOWLEDGMENTS
The authors would like to thank T. Umemiya and K. Azuma for
their supportive technical assistance. The authors were
supported by Grants-in-Aid from the Japanese Ministry of
Education, Science and Culture (grant no. 09670176).
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