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Distinguishing Intestinal Lymphoma From Inflammatory Bowel Disease in Canine Duodenal Endoscopic
Biopsy Samples
V. Carrasco, A. Rodríguez-Bertos, F. Rodríguez-Franco, A. G. Wise, R. Maes, T. Mullaney and M. Kiupel
Vet Pathol published online 8 December 2014
DOI: 10.1177/0300985814559398

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 Veterinary Pathology OnlineFirst, published on December 8, 2014 as doi:10.1177/0300985814559398

Original Article
Veterinary Pathology
1-8
Distinguishing Intestinal Lymphoma From ª The Author(s) 2014
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Inflammatory Bowel Disease in Canine DOI: 10.1177/0300985814559398
vet.sagepub.com
Duodenal Endoscopic Biopsy Samples

V. Carrasco1, A. Rodrı́guez-Bertos1,2, F. Rodrı́guez-Franco1,

A. G. Wise3, R. Maes3, T. Mullaney3, and M. Kiupel3

Abstract
Inflammatory bowel disease (IBD) and intestinal lymphoma are intestinal disorders in dogs, both causing similar chronic digestive
signs, although with a different prognosis and different treatment requirements. Differentiation between these 2 conditions is
based on histopathologic evaluation of intestinal biopsies. However, an accurate diagnosis is often difficult based on histology
alone, especially when only endoscopic biopsies are available to differentiate IBD from enteropathy-associated T-cell lymphoma
(EATL) type 2, a small cell lymphoma. The purpose of this study was to evaluate the utility of histopathology; immunohistochem-
istry (IHC) for CD3, CD20, and Ki-67; and polymerase chain reaction (PCR) for antigen receptor rearrangement (T-cell clonality)
in the differential diagnosis of severe IBD vs intestinal lymphoma. Endoscopic biopsies from 32 dogs with severe IBD or intestinal
lymphoma were evaluated. The original diagnosis was based on microscopic examination of hematoxylin and eosin (HE)–stained
sections alone followed by a second evaluation using morphology in association with IHC for CD3 and CD20 and a third evalua-
tion using PCR for clonality. Our results show that, in contrast to feline intestinal lymphomas, 6 of 8 canine small intestinal lym-
phomas were EATL type 1 (large cell) lymphomas. EATL type 2 was uncommon. Regardless, in dogs, intraepithelial lymphocytes
were not an important diagnostic feature to differentiate IBD from EATL as confirmed by PCR. EATL type 1 had a significantly
higher Ki-67 index than did EATL type 2 or IBD cases. Based on the results of this study, a stepwise diagnostic approach using
histology as the first step, followed by immunophenotyping and determining the Ki67 index and finally PCR for clonality, improves
the accuracy of distinguishing intestinal lymphoma from IBD in dogs.

Keywords
clonality, canine, immunophenotyping, Ki-67, inflammatory bowel disease, intestinal lymphoma, CD3, enteropathy-associated
T-cell lymphoma, PCR for antigen receptor rearrangement

Inflammatory bowel disease (IBD) and intestinal lymphoma
are common intestinal disorders, causing chronic persistent or
intermittent gastrointestinal clinical signs such as diarrhea,
vomiting, or weight loss in dogs. Unfortunately, the clinical
presentation of the 2 entities is often similar, and diagnostic
tests addressing those clinical signs, including fecal examina-
tion or deworming, blood tests, imaging, and correction of diet-
ary errors, often remain inconclusive.53,26,37
Morphologic assessment of the small intestine by endoscopic
or surgical biopsy remains the ultimate diagnostic tool to differ-
entiate IBD from intestinal lymphoma.26,38 While endoscopy is
less invasive and has minimal side effects for the patient in most
cases, examination is limited to the mucosa, and even some deep
mucosal lesions may escape detection in superficial biop-
sies.14,35,57 Occasionally, an intense inflammatory infiltrate
induced by the tumor may mask neoplastic cells, resulting in
an inaccurate diagnosis of IBD.37 While surgical biopsies are
guided by gross examination of the intestinal tract, and the ileo-
cecal junction as a primary location of intestinal lymphoma is
easily accessible, selection of endoscopic biopsy sites is limited
to the upper portion of the small intestine. Last, the quality of
endoscopic biopsies can be dramatically affected by collection
techniques, which prompted a recent publication of standards for
collecting endoscopic intestinal biopsies and their microscopic
1
Department of Animal Medicine and Surgery, Veterinary Medical Teaching
Hospital, Complutense University of Madrid, Avda. Puerta de Hierro s/n,
Madrid, Spain
2
Health Surveillance Centre (VISAVET), Complutense University of Madrid.
Avda. Puerta de Hierro s/n, Madrid, Spain
3
Diagnostic Center for Population and Animal Health, Beaumont Road, Lan-
sing, MI, USA
Supplemental material for this article is available on the Veterinary Pathology
website at http://vet.sagepub.com/supplemental.
Corresponding Author:
A. Rodrı́guez-Bertos, Department of Animal Medicine and Surgery, Veterinary
Teaching Hospital, Complutense University of Madrid, Avda. Puerta de Hierro
s/n, Madrid, 28040, Spain.
Email: arbertos@vet.ucm.es

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2 Veterinary Pathology
interpretation.13,55 Moreover, substantial inconsistencies in
interpretation of intestinal biopsy specimens among pathologists
were identified in some studies, and a standardization of micro-
scopic interpretations of intestinal biopsies was proposed.56,58
Based on recent advances in the classification of lymphomas
in cats, the most common primary intestinal lymphoma is
enteropathy-associated T-cell lymphoma (EATL) type 2, a small
cell lymphoma in which the cells might be morphologically
indistinguishable from IBD.14,39,43,54,59 Differentiation of IBD
from EATL type 2 may be further complicated by the potential
transformation of IBD to lymphoma, as suggested by
some.12,24,28 Other less common intestinal lymphomas in cats
include EATL type 1, a large cell lymphoma, or B-cell lympho-
mas, either diffuse large B-cell lymphomas or mucosa-
associated lymphoid tissue lymphomas. Similar to cats, a predo-
minance of intestinal T-cell lymphomas has also been reported
in dogs,21,38,40,44 which is in contrast to canine multicentric lym-
phomas as well as human gastrointestinal lymphomas that are
more commonly of B-cell origin.* Subclassification of canine
intestinal T-cell lymphomas into EATL type 1 or 2 has not been
reported. One study reported that 75% of canine intestinal
lymphomas were T-cell lymphomas that exhibited epitheliotrop-
ism, which may suggest a high incidence of EATL type 2.11
Immunohistochemistry (IHC) and clonality testing by poly-
merase chain reaction (PCR) for antigen receptor rearrange-
ment (PARR) have been used in combination with
histopathology to enhance our ability to accurately differentiate
between IBD and intestinal lymphoma in dogs, cats, and
humans.y However, the incidence of EATL type 1 and 2 in dogs
remains unclear, as does the diagnostic utility of IHC and
PARR for differentiating IBD from intestinal lymphoma in
dogs. One study evaluated this method for the diagnosis of
12 canine intestinal lymphomas and found a sensitivity of only
66.7%. Detailed studies to further differentiate canine intestinal
lymphomas and to assess the utility of IHC and PARR for dif-
ferentiating IBD from lymphoma are lacking.
The Ki-67 index, an indicator for the growth fraction of a
cell population, has been used as a prognostic marker in canine
and human lymphoma.5,18,27,34,41 It was found useful for distin-
guishing between benign and malignant lymphoproliferative
disorders in humans.6 The Ki-67 index in T cells in the lamina
propria in intestinal tissue from children with active IBD is
very low compared with that of intestinal lymphoma.15 There
are few studies concerning Ki-67 expression in canine intest-
inal T-cell lymphoma, reporting a wide range of immunoex-
pression in 3.5% to 52.6% of neoplastic cells.40 However, the
Ki-67 index has not been studied as a marker for differentiating
IBD from intestinal lymphoma in dogs.
The goal of this study was to evaluate the utility of histo-
pathology, immunophenotyping, Ki-67 index, and PARR to
more accurately diagnose intestinal lymphoma in dogs and to
differentiate it from IBD.
*References 1, 19, 23, 25, 32, 36, 42, 46, 52, 59.
y
References 7, 8, 11, 22, 24, 29, 31, 33, 39, 48, 51.
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Materials and Methods
Case Selection and Clinical History
Thirty-two dogs that had been presented to the Gastroenterol-
ogy and Endoscopy Unit of the Veterinary Medical Teaching
Hospital (Complutense University of Madrid) between 1999
and 2011 were included in this retrospective study. All dogs
had a history of chronic gastrointestinal disease. Only endo-
scopic samples from the duodenum of dogs with a morphologi-
cal diagnosis of either severe IBD (lymphoplasmacytic
enteritis) or intestinal lymphoma were included. Dogs with
parasitic disease or systemic disease affecting the intestinal
tract or dogs with exocrine pancreatic insufficiency were
excluded. Only endoscopic biopsies that were evaluated as
‘‘adequate’’ according to the recently published World Health
Organization (WHO) criteria were included in the study.57,58
History, physical examination, blood tests (hematology and
blood biochemical), abdominal ultrasound, upper endoscopy,
and endoscopic biopsy had been performed in all cases.
Histopathology, Immunohistochemistry, and PARR
Testing
Multiple biopsy samples (8–10) were obtained by endoscopy of
the duodenum of the 32 dogs. All samples were fixed in forma-
lin, routinely processed, and embedded in paraffin. Serial sec-
tions from paraffin tissue blocks were cut for routine
hematoxylin and eosin (HE) staining, IHC, and PARR.
IHC for Ki-67, CD3, and CD20 was performed, using a
streptavidin-biotin-peroxidase complex method according to
published methods.10,34 Only cells with membranous and cyto-
plasmic expression of CD3 or CD20 were evaluated as posi-
tive, and labeling of over 50% of target cells was judged as
positive. The Ki-67 index was determined by counting positive
and negative lymphocyte nuclei in 10 representative fields of
each slide (40 magnification) using computer image analysis.
The Ki-67 index (KI) was defined as the percentage of Ki-67–
positive lymphoid cells in 10 representative fields.
For PARR testing, DNA extraction was performed on five
6-mm-thick serial sections from formalin-fixed, paraffin-
embedded tissues. The rearrangement of the TCRg variable
region was assessed by amplifying the complementary
determining region 3 (CDR3) as previously described with slight
modifications.8 All PCR reactions were run in duplicate. Post-
amplification analysis of the products was performed on the
QIAxcel Advanced Instrument (Qiagen, Valencia, CA), which
allows high-resolution capillary electrophoresis of amplicons
and provides electropherograms and gel images with the QIAx-
cel Screengel Software. A result was considered clonal or posi-
tive, as well as most consistent with a neoplastic cell population,
when 1 or 2 sharp gel bands or sharp electropherogram peaks, 3
times the size of background peaks, appeared in duplicate sam-
ples within a target size range of 80 to 100 bp.8 Polyclonal
amplification, consistent with a nonneoplastic process, such as
inflammation, was identified by the presence of a broad smeared
Carrasco et al
band or multiple small peaks. Additional details on the IHC and
PARR methodology are provided as supplemental materials.
Case Evaluation
Histologic sections of small intestine from each dog were eval-
uated by 2 pathologists together (MK, AR), without knowledge
of the case history, and diagnosis was by consensus. A diagnosis
of IBD, intestinal lymphoma, or lymphoma-suspect was made
based on the microscopic appearance of the most severely
affected section. Briefly, diagnosis of IBD (lymphocytic-
plasmacytic enteritis) was based on the presence of persistent
clinical signs, lasting more than 3 weeks, and a diffuse lympho-
plasmacytic inflammatory infiltrate as the main histologic find-
ing in the endoscopic biopsies. IBD diagnosis was only
considered once all other possible causes of enteritis/infiltrates
were investigated and excluded.13,14,28,29 A diagnosis of lym-
phoma was based on a dense monomorphic population of neo-
plastic lymphocytes with signs of malignancy, such as
invasion of the muscularis mucosae, architecture distortion, or
abnormal cell size. The diagnosis of lymphoma-suspect was
based on the presence of a dense focal or patchy monomorphic
mucosal infiltrate and/or the presence of intraepithelial lympho-
cytes forming plaques or nests in 1 or more intestinal villi.
Without prior knowledge of these results, HE sections were
then studied in conjunction with IHC for CD3 and CD20 by the
same pathologists, and a second diagnosis was made. Briefly,
IBD was diagnosed based on mixed inmunophenotype with
CD3þ and CD20þ cells, T-cell lymphoma based on large num-
bers of CD3þ cells with scarce CD20þ cells corresponding to the
inflammatory background, B-cell lymphoma based on numerous
CD20þ cells with sparse CD3þ cells corresponding to the
inflammatory background, or lymphoma-suspect based on focal
dense areas of CD3þ cells in the villi and/or CD3þ intraepithelial
plaques. The diagnoses were revised based on uniformity of cell
labeling, phenotype in association with cell localization, and cell
size as well as growth pattern. The number of cases that were
reclassified after immunophenotyping was recorded. Each case
was analyzed a third time, without knowledge of previous
diagnosis, by combining histomorphology (HE), IHC, and
PARR. The differences in diagnosis at each step were compared.
In addition, morphologic features of crypt distension (normal/
mild/moderate/marked), lacteal dilation (normal/mild/moderate/
marked), villous stunting (normal/mild/moderate/marked),
mucosal fibrosis (normal/mild/moderate/marked), presence of
increased intraepithelial lymphocytes (more than 20–30 per
40 stretch of villous epithelium),13 the distribution of the lym-
phocytic infiltrate (focal, multifocal or diffuse), and monomorph-
ism versus polymorphism of lymphocytes based on nuclear size
and morphology were recorded for each case. Furthermore,
intraepithelial lymphocytes were assessed in the entire sample
and defined as ‘‘diffuse’’ or ‘‘involving one to several villi.’’34
The infiltrates of T cells within the epithelium were recorded as
either surface or crypt infiltrates or both, and intraepithelial infil-
trates were divided into single cells, nests, or plaques. Nests of
lymphocytes were defined as 5 clustered intraepithelial
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3
lymphocytes, while plaques were defined as 5 adjacent epithe-
lial cells overrun by lymphocytes.33 In the lymphoma group, the
number of mitotic figures per ten 400 fields was counted, and
cell size (large vs small, >1.5 blood cell size vs <1.5 blood cell
size) was evaluated.49 The lymphoma cases were classified
according to the WHO classification of lymphoid neoplasms.3,47
Statistical Analysis
Data were analyzed by using SPSS statistical software (SPSS,
Inc., an IBM Company, Chicago, IL). A ‘‘Descriptive statis-
tics’’ procedure was used for quantitative variables and a ‘‘fre-
quencies’’ procedure for qualitative variables.16 The Pearson
w1 test was used to determine independency between quantita-
tive variables. One-way analysis of variance (ANOVA) was
used for mean comparison of Ki-67 among groups. P  .05 was
considered statistically significant.
Results
Clinical Data
The age of the dogs in this study was 7.65 + 3.00 years (mean
+ SD). Twenty dogs (62.5%) were male and 12 (37.5%) were
female. The most represented breeds were Rottweiler (n ¼ 5),
Yorkshire terrier (n ¼ 4), boxer (n ¼ 3), cocker spaniel (n ¼ 3),
and gos d’Atura catalán (n ¼ 2).
Clinical signs at presentation were related to the gastroin-
testinal tract and included weight loss (96.9%), chronic diar-
rhea of small bowel origin (81.3%), and chronic vomiting
(53.1%). A remarkable number of cases also had varying
degrees of decreased appetite (53.1%).
Case Evaluation
All 32 cases were diagnosed by histopathology, immunopheno-
typing, and PARR. Based on morphologic evaluation alone, 7
cases were classified as intestinal lymphoma (21.9%), 12 cases
as IBD (37.5%), and 13 as lymphoma-suspect (40.6%). When
IHC for CD3 and CD20 was analyzed in conjunction with the
microscopic findings, 8 cases were classified as T-cell lym-
phoma (25%; Fig. 2), 15 cases as IBD (46.9%; Fig. 1), and 9
cases as lymphoma-suspect (28.1%). There were no B-cell
lymphomas. IHC allowed reclassification of 6 of the 13 cases
originally classified as lymphoma-suspect; 5 were thought to
be IBD and 1 a T-cell lymphoma. However, 2 cases originally
diagnosed as IBD were reclassified as lymphoma-suspect
based on the combined HE and IHC results. All 7 cases origi-
nally diagnosed as lymphoma remained in the lymphoma group
and were further classified as T-cell lymphomas. When PARR
results were used in conjunction with HE and IHC results, all
9 cases that had been included in the lymphoma-suspect group
were diagnosed as IBD, all 15 cases in the IBD group were
diagnosed as IBD, and 8 cases of lymphoma were diagnosed
as T-cell lymphoma (Tables 1, 2). All lymphomas in this study
were T-cell lymphomas. The neoplastic lymphocytes were
classified as large cells in 6 of these cases and as small cell
4 Veterinary Pathology

Figure 1. Inflammatory bowel disease, small intestine, dog. (a) Hematoxylin and eosin (HE), (b) immunohistochemistry (IHC) for CD3, and (c) immu-
nohistochemistry for CD20. Notice the mixed immunophenotype, characteristic of an inflammatory process. Figure 2. Intestinal T-cell lymphoma,
small intestine, dog. (a) HE, (b) IHC for CD3, and (c) IHC for CD20. Figure 3. Inflammatory bowel disease (lymphoma-suspect), small intestine, dog.
Intraepithelial plaques (5 adjacent epithelial cells overrun by lymphocytes) in the surface epithelium: pattern of intraepithelial T cells depicted in (a) HE
and (b) IHC for CD3. Figure 4. Inflammatory bowel disease, small intestine, dog. Ki-67 immunolabeling of 10.75%. Figure 5. Intestinal T-cell lym-
phoma, small intestine, dog. Ki-67 immunolabeling of 60.07% at lower (a) and higher (b) magnification. Figure 6. T-cell receptor gamma
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Carrasco et al
in 2 cases, resulting in a diagnosis of EATL type 1 and type 2,
respectively.3,47 The average number of mitosis per 400 field
was 13.8 for EATL type 1 and 7.5 for EATL type 2.
PARR Testing
PARR testing was performed on all 32 cases. Six cases were
determined to have a clonal population of T cells, while the rest
of the samples were polyclonal. All clonal samples had been
diagnosed as T-cell lymphoma based on HE and IHC results.
However, 2 samples from the lymphoma group were polyclo-
nal based on PARR testing, a result that was considered incor-
rect after further review of the morphology and IHC results,
which supported the diagnoses of lymphoma (Fig. 6). When
HE and IHC results were considered in combination with
PARR, all the cases included in the IBD and lymphoma-
suspect groups were classified as IBD.
Morphologic Features
All samples had a severe lymphocytic and variable plasmacytic
infiltration within the lamina propria. The distribution of this
severe infiltration was diffuse in 20 of 24 cases of IBD and 6
of 8 cases of lymphoma and multifocal in 4 of 24 and 2 of
8 cases of IBD and lymphoma, respectively. According to The
World Small Animal Veterinary Association (WSAVA) stan-
dards,13 villous stunting was found in most cases but was sta-
tistically more severe in lymphomas (P ¼ .004). Epithelial
injury was observed more frequently and was more severe in
lymphomas than in IBD cases (P ¼ .002). Crypt distension, lac-
teal dilation, and mucosal fibrosis appeared to be similar in
cases of IBD and lymphoma.
Intraepithelial lymphocytes were always CD3 positive and
CD20 negative and were therefore evaluated in CD3-labeled
slides. Twenty-four cases (8/8 lymphoma, 16/24 IBD) had
increased intraepithelial infiltrates, with most cases presenting
with diffuse intraepithelial infiltrates within the surface epithe-
lium. Intraepithelial infiltrates involving only 1 to several villi
were observed in 2 of 8 cases of lymphoma and 4 of 24 cases of
IBD. Intraepithelial infiltrates affecting both the surface and the
crypts were significantly less common in IBD (2/24) than in lym-
phoma (5/8; P ¼ .004) cases. Of the 24 cases that had intraepithe-
lial infiltrates, 7 cases had only single intraepithelial lymphocytes
(6/24 IBD, 1/8 lymphoma), whereas 5 had nests (3/24 IBD, 2/8
lymphoma), and 12 had plaques (7/24 IBD, 5/8 lymphoma; Fig. 3).
In 15 cases (8/8 lymphoma, 7/24 IBD), the lymphocytic
infiltration was predominantly monomorphic. Monomorphic
infiltrates in IBD cases were observed in the sections most
severely affected, and this morphologic feature had originally
led to a diagnosis of lymphoma-suspect in all 7 IBD cases. A
Figure 6. (continued) (TCRG) rearrangements in intestinal biopsy samples from dogs with intestinal lymphoma or inflammatory bowel
disease. All samples were run in duplicate. (a) Electropherogram: polyclonal. (b) Electropherogram: monoclonal case (89-pb monoclonal
peak can be noted). (c) Electrophoresis gel, lanes A1 and A2: monoclonal case (sharp monoclonal band can be identified). Lanes A3 and
A4: polyclonal case (smeared band is evident).
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5
diagnosis of IBD was finally reached based on PARR testing.
Polymorphic infiltrates were identified in 17 cases of IBD.
Immunohistochemistry for Proliferation Index Ki-67
IHC for Ki-67 was performed on all 32 cases. All lymphomas
expressed a significantly higher Ki-67 index (median + SD:
52.9% + 11.9%) than IBD (15.3% + 10.0%) (P < .001) cases.
Ki-67 immunolabeling in lymphomas ranged from 30% to 64%
of neoplastic cells, while only 5 cases of IBD had a Ki-67 index
higher than 20%, and only 2 had a Ki-67 index between 30%
and 50% (Figs. 4 and 5).
Discussion
Similar to previous studies, clinical signs or the gross appear-
ance of the duodenal surface as observed by endoscopy did not
correspond to the degree of histopathologic changes and were
insufficient for differentiating between intestinal lymphoma and
severe IBD in this study.14,28,37,43,45 While surgical biopsies pro-
vide the most diagnostic detail, histologic interpretation of duo-
denal tissues obtained by endoscopy is difficult, and ideally,
sections throughout the small intestine are encouraged.14,37,56
The examined morphologic features such as villous stunting,
crypt distension, lacteal dilation, or mucosal fibrosis did not help
differentiate between severe IBD and lymphoma.13 While dogs
with intestinal lymphoma would be expected to present with a
monomorphic lymphocytic population, in many cases, intestinal
lymphomas may occur in conjunction with lymphoplasmacytic
inflammation.37 Combining HE with IHC evaluation improved
the ability to differentiate canine intestinal T-cell lymphomas
from inflammation, similar to what has been reported in
cats.2,34,54 In our study, all lymphoma cases were diffuse T-
cell lymphomas with a lymphoplasmacytic inflammatory back-
ground, which is similar to an earlier study.38 Other studies iden-
tified between 63% and 75% of gastrointestinal lymphomas as
having a T-cell phenotype, but these studies also examined the
stomach, which is known to have a higher incidence of B-cell
lymphomas in dogs and cats.4,11,20,39,44
The only statistically significant morphologic parameter for
differentiating IBD from lymphoma was the presence of intrae-
pithelial infiltrations in both surface and crypt epithelium. In
contrast to cats, a significant number of dogs with a diagnosis
of IBD based on the combined HE, IHC, and PARR results
exhibited marked intraepithelial infiltrates. The occurrence of
intraepithelial plaques and nests that has been shown to be a
strong indicator of EATL type 2 in cats was not predictive of
intestinal lymphoma in dogs. Since EATL type 1 was much
more commonly observed in dogs than EATL type 2, epithelio-
tropism may be a much less important diagnostic parameter in
dogs. In humans, EATL type 1 is the predominant intestinal
6 Veterinary Pathology

Table 1. Comparison of Diagnoses Based on Morphologic Evaluation capillary electrophoresis rather than a polyacrylamide gel,
Alone (HE), in Combination With Immunophenotyping (IHC), and in leading to an increased resolution and thereby sensitivity. We
Combination With Immunophenotyping and PARR. performed PARR testing in duplicate to ensure that no single
HE HE þ IHC HE þ IHC þ PARR bands representing pseudoclones were misinterpreted as a clo-
nal cell population. In contrast to previous studies, we also ana-
IBD 12 15 24 lyzed both not applicable native and denatured-reannealed
Lymphoma-suspect 13 9 0 products (heteroduplex analysis).31 Denaturation and reanneal-
Lymphoma 7 8 8
ing do not affect bands amplified from truly clonal cells but
B-cell lymphoma — 0 0
T-cell lymphoma — 8 8 will produce a smear for pseudoclonal cell populations. Failure
EATL type 1 — 2 2 to eliminate pseudoclones may result in loss of specificity of up
EATL type 2 — 6 6 to 10%.31,50 Integrating the newly developed primers into
PARR testing combined with morphology and IHC will likely
EATL, enteropathy-associated T-cell lymphoma; HE, hematoxylin and eosin;
IBD, inflammatory bowel disease; IHC, immunohistochemistry; PARR, poly-
aid in making a more accurate diagnosis.
merase chain reaction for antigen receptor rearrangement; —, not applicable. Ki-67 has been used as a marker of cellular proliferation in
canine and human lymphoma, and the Ki-67 index correlates
with the prognosis of canine and human lymphoma.5,19,34,41
Table 2. Changes of Diagnosis After Combining the Morphologic
Evaluation With Immunophenotyping and With Immunophenotyping
There are no previous studies investigating the utility of the
Plus PARR. Ki-67 index to differentiate canine IBD from intestinal lym-
phoma. In humans, lamina propria T-lymphocytes in IBD are
HE þ IHC þ normally not dividing, despite mucosal inflammation.15
Change of diagnosis HE þ IHC PARR Furthermore, benign versus malignant lymphoid proliferations
IBD ! lymphoma suspect 2 0 have been differentiated based on the Ki-67 index in lymph
IBD ! T-cell lymphoma 0 0 nodes.6 We demonstrated that the Ki-67 index was signifi-
Lymphoma suspect ! IBD 5 9 cantly higher in canine lymphomas than in IBD. Lymphomas
Lymphoma suspect ! T-cell 1 0 exhibited between 30% and 62% of Ki-67–positive cells, while
lymphoma most cases of IBD displayed a Ki-67 index lower than 25%.
Lymphoma ! IBD 0 0 Previous studies indicated that canine T-cell lymphomas may
Lymphoma ! lymphoma suspect 0 0
have a higher Ki-67 index than B-cell lymphomas.17,42 The
HE, hematoxylin and eosin; IBD, inflammatory bowel disease; IHC, mean Ki-67 index in the lymphoma group was higher than pre-
immunohistochemistry; PARR, polymerase chain reaction for antigen receptor viously published values for intestinal lymphomas40 but similar
rearrangement.
to multicentric T-cell lymphomas in other studies.17,42 EATL
type 2 lymphomas had the lowest Ki-67 index, and a similar
T-cell lymphoma, with an 80% to 90% prevalence, while in index may be observed in IBD cases with large numbers of
cats, EATL type 2 is the predominant intestinal T-cell lym- infiltrating T cells. In the current study, only 1 case of IBD had
phoma.3,34,39,47 Our data are similar to a previous study that a high Ki-67 index (47.15%), similar to the Ki-67 index in lym-
reported 3 cases of small cell, 4 of intermediate cell, and 4 of phomas. The affected dog did not respond to treatment for sev-
large cell T-cell lymphomas.40 eral weeks and was euthanized. No follow-up necropsy was
Even when combining morphologic evaluation with IHC available, and it remains unclear whether this dog had intestinal
and PARR, a certain diagnosis could not be reached in all lymphoma. Based on our current knowledge, Ki-67 should not
cases. Some of the cases in our study that were diagnosed as be used as a sole parameter to differentiate IBD from intestinal
IBD based on polyclonal PARR testing may represent early lymphoma. However, integration of the Ki-67 index into a
stages of EATL type 2.7,9,47,50 A polyclonal PARR result of diagnostic panel composed of the histopathologic evaluation,
an intestinal lymphoma may be due to low numbers of neoplas- immunophenotyping, and PARR testing can be useful. These
tic cells in a strong inflammatory background or low sensitivity tests should be performed in sequence, and test results should
of the multiplex PCRs covering insufficient antigen receptor only be interpreted in context. Especially for dogs that have
rearrangements. A recent publication of new sets of primers for been diagnosed as lymphoma suspects or with IBD but do not
PARR for canine TCRG improved the test sensitivity in dogs respond appropriately to treatment, the histopathology should
by 25% compared with the primers used in this study 30 Con- be reviewed in combination with immunophenotyping, PARR
sidering that 2 cases diagnosed as lymphoma based on mor- testing, and evaluation of the Ki-67 index. Immunophenotyp-
phology and IHC results were polyclonal by PARR testing, a ing and determining the Ki-67 index are recommended in any
sensitivity of less than 75% for PARR also has to be assumed case with a morphologic diagnosis of lymphoma, and PARR
for our study. The sensitivity of PARR for detecting clonal cell testing can be performed as a confirmatory test. Regardless,
proliferations was lower than what has been published for other additional biopsies may be required throughout the course of
types of canine lymphoma8 but higher than previously reported the disease. This systematic assessment of canine endoscopic
by others (66.7%) for canine intestinal lymphoma.22 The biopsies will increase diagnostic accuracy and will enable both
increase in sensitivity in our study may be due to the use of proper prognosis and appropriate treatment.

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Carrasco et al
Author Contribution
Conception or design: VC, ARB, FRF, AW, RM, TM, MK. Data
acquisition, analysis, or interpretation: VC, ARB, FRF, AW, RM,
TM, MK. Drafting the manuscript: VC, MK. All authors participated
in critically revising the manuscript, gave final approval, and agree to
be accountable for all aspects of work to ensure integrity and accuracy.
Acknowledgements
We thank Tom Wood and the DCPAH Histology and Immunohisto-
chemistry Laboratory. We also thank the personnel of the DCPAH
Virology Laboratory. We are grateful to the Complutense Veterinary
Teaching Hospital Histology Laboratory for technical support and to
Santiago Cano for his help with statistics.
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
Violeta Carrasco’s stay in the DCPH (Michigan State University) was
financed by the scholarship program of the Spanish Ministry of Educa-
tion: University Teaching Training Program (Formación de Profesorado
Universitario-FPU), code AP2007-01210.
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