Brief Communication

Veterinary Pathology
1-4
Coexpression of CD3 and CD20 in Canine ª The Author(s) 2018
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Enteropathy-Associated T-cell Lymphoma DOI: 10.1177/0300985817747326
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Erica L. Noland1 and Matti Kiupel1

Abstract
The majority of primary intestinal lymphomas in dogs are T-cell lymphomas, with enteropathy-associated T-cell lymphoma (EATL)
large cell type (type 1) being the most common. While most T-cell lymphomas express the T-cell marker CD3, there is increasing
evidence that some human and canine T-cell lymphomas coexpress the B-cell marker CD20. We describe 3 cases of CD3þ,
CD20þ, Pax5- EATL type 1 in dogs. All 3 cases had clonal rearrangement of T-cell receptor gamma. Initial clinical signs included
weight loss, inappetence, diarrhea, and/or vomiting. The mean age was 9 years (range 3–12). Survival was highly variable ranging
from 20 days to longer than 1.6 years. Considering the different chemotherapeutic response of T-cell versus B-cell lymphomas,
accurate diagnosis of lymphomas coexpressing CD3 and CD20 as EATL type 1 based on histologic features and clonality results is
important. Regardless, the clinical and/or prognostic significance of neoplastic T cells expressing CD20 is unclear.

Keywords
CD3, CD20, PAX5, immunohistochemistry, PCR for antigen receptor rearrangement, enteropathy-associated T-cell lymphoma,
EATL

Historically, the vast majority of primary intestinal lympho-
mas in dogs have been reported as peripheral T-cell lympho-
mas, not otherwise specified.3 While feline intestinal T-cell
lymphomas have been well characterized and, similar to their
human counterparts, have been subclassified according to cell
morphology and pattern by the World Health Organization
(WHO) as enteropathy-associated T-cell lymphoma (EATL)
large cell (type 1) and small cell (EATL type 2);7,8,13 this
advance in classification has only recently been established
in dogs.2,8,13 In contrast to feline intestinal T-cell lymphomas,
which are predominately EATL type 2 and characterized as a
monomorphic population of small T lymphocytes often form-
ing variably sized intraepithelial nests and plaques, canine
intestinal T-cell lymphomas are most commonly reported as
large cell type with or without accompanying inflammation
and/or epitheliotropism or, according to human classification,
EATL type 1.2,8,13
In most intestinal T-cell lymphomas, neoplastic cells are
expected to express the T-cell marker CD3 and are not immu-
noreactive for the B-cell marker CD20. However, there is an
increasing body of literature of human peripheral T-cell lym-
phomas and fewer cases of cutaneous epitheliotropic T-cell
lymphomas that coexpress CD20.4,11 Recently, coexpression
of CD3 and CD20 was reported in a cutaneous epitheliotropic
T-cell lymphoma (mycosis fungoides) in a single dog.1 While
the clinical significance has yet to be determined in either
species, recognition of such dual expression has important
diagnostic implications that ultimately guide the therapeutic
approach. To our knowledge, this is the first report of
coexpression of CD3 and CD20 in primary intestinal T-cell
lymphoma in dogs.
Three dogs were included in this case series. Tissues
received included 3 and 4 small endoscopically biopsied sec-
tions of small intestinal mucosa from dogs 1 and 2, respec-
tively, and 2 full thickness small intestinal biopsies from dog
3. Surgical biopsy specimens had been routinely fixed in for-
malin and embedded in paraffin. From each case, 5 mm serial
sections were routinely stained with hematoxylin and eosin.
CD3, CD20, and Pax5 immunohistochemistry, including dual
labeling for CD3 and CD20 as well as dual labeling for CD3
and Pax5 were performed as previously described.1,2 Polymer-
ase chain reaction (PCR) for antigen receptor rearrangement
(T-cell clonality) was performed on each case as previously
described.2
Dog 1 was a 12-year-old, male castrated Cocker Spaniel 
Poodle that had weight loss, inappetence, and diarrhea for sev-
eral months duration. Endoscopy showed a rough small intest-
inal mucosa, and small intestinal mucosal biopsies were taken.
Dog 2 was a 12-year-old, female spayed Pembroke Welsh Corgi
1
Department of Pathobiology and Diagnostic Investigation, Veterinary Diag-
nostic Laboratory, Michigan State University, Lansing, MI, USA
Corresponding Author:
Matti Kiupel, Department of Pathobiology and Diagnostic Investigation,
Veterinary Diagnostic Laboratory, Michigan State University, 4125 Beaumont
Rd, Lansing, MI 48910, USA.
Email: kiupel@dcpah.msu.edu
2 Veterinary Pathology XX(X)
that had intermittent soft stool for 1 year duration and diarrhea
with occasional hematochezia for approximately 4 months dura-
tion. The dog was treated with metronidazole and a change in
diet to bland home cooked meals. The dog was then transitioned
off metronidazole and onto tylosin powder. Endoscopy showed a
diffusely hyperemic, friable, and granular small intestinal
mucosa, and duodenal mucosal biopsies were taken. Dog 3 was
a 3-year-old, male American Stafford Terrier that had vomiting
and inappetence for 5-6 days duration, and an acute onset of
lethargy, diarrhea, and inability to walk. The dog was febrile
and had swollen joints, and abdominal exploratory surgery
revealed a ruptured mass at the ileocecal junction, septic abdo-
men, and mesenteric lymphadenopathy. The mass and an
enlarged cecal lymph node were resected.
On histopathologic evaluation, in mucosal tissue sections
from dog 1, there were large lymphocytes moderately expand-
ing the villar mucosal lamina propria and forming multifocal
intraepithelial nests and plaques (Figs. 1–2). In mucosal tissue
sections from dog 2, there was a monomorphic population of
intermediate-sized lymphocytes with a similar distribution as
noted in dog 1. In full thickness tissue sections from dog 3,
there was a transmural infiltrate of monomorphic large lym-
phocytes arranged in sheets that extended into the adjacent
mesenteric adipose tissue.
In all cases, the neoplastic lymphocytes had strong perimem-
branous labeling for CD3 (Fig. 3a). In addition, the majority of
neoplastic lymphocytes, including those noted within the muco-
sal epithelium, had perimembranous labeling for CD20 of vari-
able intensity (Figs. 3–4). These neoplastic lymphocytes were
not immunoreactive for Pax5 (Fig. 5). PARR testing showed
clonal rearrangement of the T-cell receptor gamma gene for all
cases. Since the neoplastic lymphocytes in all 3 cases were
negative for another B-cell marker, Pax5, and monoclonal rear-
rangement of the T-cell antigen receptor gene was confirmed
for each case, a diagnosis of a CD3þ, CD20þ enteropathy-
associated T-cell lymphoma, large cell type (EATL type 1) was
made for each dog.
For dog 1, prednisone therapy initially decreased clinical
signs; however, at 20 days after the molecular confirmation
of a lymphoma diagnosis, the animal developed weakness,
profuse hemorrhagic diarrhea, vomiting, and inappropriate uri-
nation and subsequently developed increased respiratory effort
and a tense painful abdomen. Due to a poor prognosis, eutha-
nasia was elected at that time.
For dog 2, following the start of tylosin therapy and at
10 days after the molecular confirmation of a lymphoma diag-
nosis, the animal had not had any diarrhea. While the animal
has not been examined since that time, the dog was reportedly
alive at 1.6 years after the initial diagnosis.
For dog 3, ultrasound examination at 26 days postsurgery
showed no signs of recurrence or progression of disease. At 96
days postsurgery, the dog had pain while defecating, and an
ultrasound examination showed an 8.5 cm jejunal mass. The
jejunal mass was debulked, and a modified T-cell multiagent
chemotherapy protocol, including asparaginase, vincristine,
lomustine, and prednisone, was started 8 days later. Chemotherapy
was continued on a weekly basis in an alternating fashion.
Progressive disease was noted on ultrasound examination at
159 days postdiagnosis. An abdominal exploratory surgery
was performed at 181 days postdiagnosis, and the dog was
euthanized due to complications and advanced stage disease
throughout the abdomen.
CD20 is typically considered a marker of B-cell lineage.
Unlike Pax5, which was negative in these cases and is
expressed throughout the entirety of B-cell maturation, CD20
is normally expressed on the surface of B lymphocytes begin-
ning at the pre-B cell stage before immunoglobulin m heavy
chains develop in the cytoplasm, and expression persists up to
terminal plasma cell differentiation.11 This transmembrane
phosphoprotein has unconfirmed roles in both calcium trans-
port and T-cell-independent antigen-induced activation of B
cells.11 However, in normal peripheral blood of humans, a
subset of T lymphocytes with low expression of CD20 has been
detected by flow cytometry, which are known as CD20dim T
lymphocytes.10 These CD20dim T cells are phenotypically dif-
ferent from CD20- T cells and are more likely to be CD8,
CD45RO, and g/d T-cell antigen receptor positive; however,
their function remains unknown.5
It is unclear whether CD20 expression in this subset of canine
T-cell lymphomas reflects aberrant expression of CD20 or
whether these cells originated from a subpopulation of normal
CD20dim T cells, and if either of these possibilities have any
clinical relevance.10–12 A third theory involves false-positive
labeling of the neoplastic T cells due to cross-reactivity of the
anti-CD20 antibody.12 This theory is less likely because the
observed CD20 labeling in T cells was limited to neoplastic T
cells only and not inflammatory T cells at greater distance from
the lesion, and because such dual expression is rare in our routine
biopsy service.
Similar CD20 expression has been recognized in a small
percentage of human T-cell lymphomas and in a single case of
a canine cutaneous epitheliotropic lymphoma.1,4,11 In humans,
reports include 2 descriptions of enteropathy-associated T-cell
lymphoma with CD20 expression, one of which was initially
misdiagnosed as B-cell lymphoma.6,11 The prognostic and ther-
apeutic implications of such CD20 expression is unclear.
Follow-up information in the human cases was limited, but 1
of the 2 reports indicated a poor prognosis.11 Regardless, a mis-
diagnosis of a T-cell lymphoma as a B-cell lymphoma can have
serious clinical implications because T-cell lymphoma is typi-
cally more aggressive than B-cell lymphoma, resulting in a dif-
ferent prognosis and therapeutic strategy. In limited follow-up
information for this case series in dogs, the biologic behavior
was aggressive in 2 dogs and 1 dog was still alive after 1.6 years
without further clinical information. Monoclonal therapy against
CD20 has at least been shown to deplete CD20dim T lympho-
cytes in humans with multiple sclerosis, which raises the ques-
tion whether targeted therapy against CD20 may be of value for
T-cell lymphomas that have CD20 expression.9
EATL type 1 lymphomas are often associated with trans-
mural infiltration.7,8,13 In 1 case (dog 3), there was peritonitis at
the time of diagnosis due to rupture of the intestinal wall
Noland and Kiupel 3

Figures 1–2. Enteropathy-associated T-cell lymphoma (EATL) type 1, small intestine, dog 1. Figure 1. Neoplastic lymphocytes expand the
lamina propria and clustered or individualized neoplastic cells infiltrate the epithelium. Hematoxylin and eosin (HE). Figure 2. Neoplastic
lymphocytes are large, vesiculate, have 1–2 variably prominent nucleoli, and occasionally contain fine eosinophilic intracytoplasmic granules
(inset). HE. Figure 3. EATL type 1, small intestine, dog 2. Neoplastic lymphocytes have strong perimembranous to cytoplasmic labeling for CD3
(a) and, in a large percentage of this cell population, CD20 (b). Immunohistochemistry (IHC) with 3,3’-diaminobenzidine (DAB) chromogen.
Figures 4–5. EATL type 1, small intestine, dog 1. Figure 4. Neoplastic cells often label for CD3 (brown) and CD20 (red). Dual labeling IHC for
CD3 (DAB, brown), and CD20 (red). Figure 5. Neoplastic lymphocytes do not have nuclear labeling for Pax5. Rare non-neoplastic B
lymphocytes have red nuclear labeling for Pax5 (arrow). Dual labeling IHC for CD3, DAB (brown), and Pax5 (red).

secondary to full thickness invasion of the neoplasm; however, The aforementioned human case of a CD20-expressing EATL
the full depth of invasion could not be evaluated in the other 2 also had intestinal perforation.10 No neoplastic cells were
cases (dogs 1 and 2) due to submission of endoscopic biopsies. reported in the cecal lymph node in dog 3, and the oral and
4 Veterinary Pathology XX(X)
aboral margins of the resected intestinal mass were reported
free of neoplastic cells at the time of diagnosis.
This case series demonstrates the existence of CD3þ, CD20þ
enteropathy-associated T-cell lymphoma in dogs and highlights
the importance of combining histomorphology, clinical presen-
tation, immunophenotyping, and PARR testing to properly diag-
nose these neoplasms. Further, given the potential prognostic
and therapeutic implications, immunophenotyping should routi-
nely be considered for not only diagnostic purposes. Recognition
of this particular phenotype of canine EATL type 1 will allow
collection of additional cases and investigation of their biologi-
cal behavior to ultimately determine the clinical significance and
prognostic value of CD20 expression in canine enteric T-cell
lymphomas.
Acknowledgements
We thank Madison Operacz at the Michigan State University Veter-
inary Diagnostic Laboratory (MSU VDL) for performing the clonality
testing, and the histology department at MSU VDL, particularly Tom
Wood, for IHC. Gratitude to Dr James Walberg, VetPath Services,
Stone Ridge, NY; Dr Kate Vickery, Hope Vet Specialists, Malvern,
PA; Dr Julie Stanton, Cornell University Hospital for Animals, Ithaca,
MI; and Animal Emergency and Referral Associates, Fairfield, NJ for
submission of the cases and/or providing follow-up data.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, author-
ship, and/or publication of this article.
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