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

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2 Veterinary Pathology

interpretation.13,55 Moreover, substantial inconsistencies in Materials and Methods

interpretation of intestinal biopsy specimens among pathologists
were identified in some studies, and a standardization of micro- Case Selection and Clinical History
scopic interpretations of intestinal biopsies was proposed.56,58 Thirty-two dogs that had been presented to the Gastroenterol-
Based on recent advances in the classification of lymphomas ogy and Endoscopy Unit of the Veterinary Medical Teaching
in cats, the most common primary intestinal lymphoma is Hospital (Complutense University of Madrid) between 1999
enteropathy-associated T-cell lymphoma (EATL) type 2, a small and 2011 were included in this retrospective study. All dogs
cell lymphoma in which the cells might be morphologically had a history of chronic gastrointestinal disease. Only endo-
indistinguishable from IBD.14,39,43,54,59 Differentiation of IBD scopic samples from the duodenum of dogs with a morphologi-
from EATL type 2 may be further complicated by the potential cal diagnosis of either severe IBD (lymphoplasmacytic
transformation of IBD to lymphoma, as suggested by enteritis) or intestinal lymphoma were included. Dogs with
some.12,24,28 Other less common intestinal lymphomas in cats parasitic disease or systemic disease affecting the intestinal
include EATL type 1, a large cell lymphoma, or B-cell lympho- tract or dogs with exocrine pancreatic insufficiency were
mas, either diffuse large B-cell lymphomas or mucosa- excluded. Only endoscopic biopsies that were evaluated as
associated lymphoid tissue lymphomas. Similar to cats, a predo- ‘‘adequate’’ according to the recently published World Health
minance of intestinal T-cell lymphomas has also been reported Organization (WHO) criteria were included in the study.57,58
in dogs,21,38,40,44 which is in contrast to canine multicentric lym- History, physical examination, blood tests (hematology and
phomas as well as human gastrointestinal lymphomas that are blood biochemical), abdominal ultrasound, upper endoscopy,
more commonly of B-cell origin.* Subclassification of canine and endoscopic biopsy had been performed in all cases.
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-
Histopathology, Immunohistochemistry, and PARR
ism, which may suggest a high incidence of EATL type 2.11
Immunohistochemistry (IHC) and clonality testing by poly-
Testing
merase chain reaction (PCR) for antigen receptor rearrange- Multiple biopsy samples (8–10) were obtained by endoscopy of
ment (PARR) have been used in combination with the duodenum of the 32 dogs. All samples were fixed in forma-
histopathology to enhance our ability to accurately differentiate lin, routinely processed, and embedded in paraffin. Serial sec-
between IBD and intestinal lymphoma in dogs, cats, and tions from paraffin tissue blocks were cut for routine
humans.y However, the incidence of EATL type 1 and 2 in dogs hematoxylin and eosin (HE) staining, IHC, and PARR.
remains unclear, as does the diagnostic utility of IHC and IHC for Ki-67, CD3, and CD20 was performed, using a
PARR for differentiating IBD from intestinal lymphoma in streptavidin-biotin-peroxidase complex method according to
dogs. One study evaluated this method for the diagnosis of published methods.10,34 Only cells with membranous and cyto-
12 canine intestinal lymphomas and found a sensitivity of only plasmic expression of CD3 or CD20 were evaluated as posi-
66.7%. Detailed studies to further differentiate canine intestinal tive, and labeling of over 50% of target cells was judged as
lymphomas and to assess the utility of IHC and PARR for dif- positive. The Ki-67 index was determined by counting positive
ferentiating IBD from lymphoma are lacking. and negative lymphocyte nuclei in 10 representative fields of
The Ki-67 index, an indicator for the growth fraction of a each slide (40 magnification) using computer image analysis.
cell population, has been used as a prognostic marker in canine The Ki-67 index (KI) was defined as the percentage of Ki-67–
and human lymphoma.5,18,27,34,41 It was found useful for distin- positive lymphoid cells in 10 representative fields.
guishing between benign and malignant lymphoproliferative For PARR testing, DNA extraction was performed on five
disorders in humans.6 The Ki-67 index in T cells in the lamina 6-mm-thick serial sections from formalin-fixed, paraffin-
propria in intestinal tissue from children with active IBD is embedded tissues. The rearrangement of the TCRg variable
very low compared with that of intestinal lymphoma.15 There region was assessed by amplifying the complementary
are few studies concerning Ki-67 expression in canine intest- determining region 3 (CDR3) as previously described with slight
inal T-cell lymphoma, reporting a wide range of immunoex- modifications.8 All PCR reactions were run in duplicate. Post-
pression in 3.5% to 52.6% of neoplastic cells.40 However, the amplification analysis of the products was performed on the
Ki-67 index has not been studied as a marker for differentiating QIAxcel Advanced Instrument (Qiagen, Valencia, CA), which
IBD from intestinal lymphoma in dogs. allows high-resolution capillary electrophoresis of amplicons
The goal of this study was to evaluate the utility of histo- and provides electropherograms and gel images with the QIAx-
pathology, immunophenotyping, Ki-67 index, and PARR to cel Screengel Software. A result was considered clonal or posi-
more accurately diagnose intestinal lymphoma in dogs and to tive, as well as most consistent with a neoplastic cell population,
differentiate it from IBD. 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
*References 1, 19, 23, 25, 32, 36, 42, 46, 52, 59. amplification, consistent with a nonneoplastic process, such as
y
References 7, 8, 11, 22, 24, 29, 31, 33, 39, 48, 51. inflammation, was identified by the presence of a broad smeared

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Carrasco et al 3

band or multiple small peaks. Additional details on the IHC and lymphocytes, while plaques were defined as 5 adjacent epithe-
PARR methodology are provided as supplemental materials. 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
Case Evaluation size) was evaluated.49 The lymphoma cases were classified
Histologic sections of small intestine from each dog were eval- according to the WHO classification of lymphoid neoplasms.3,47
uated by 2 pathologists together (MK, AR), without knowledge
of the case history, and diagnosis was by consensus. A diagnosis Statistical Analysis
of IBD, intestinal lymphoma, or lymphoma-suspect was made
based on the microscopic appearance of the most severely Data were analyzed by using SPSS statistical software (SPSS,
affected section. Briefly, diagnosis of IBD (lymphocytic- Inc., an IBM Company, Chicago, IL). A ‘‘Descriptive statis-
plasmacytic enteritis) was based on the presence of persistent tics’’ procedure was used for quantitative variables and a ‘‘fre-
clinical signs, lasting more than 3 weeks, and a diffuse lympho- quencies’’ procedure for qualitative variables.16 The Pearson
plasmacytic inflammatory infiltrate as the main histologic find- w1 test was used to determine independency between quantita-
ing in the endoscopic biopsies. IBD diagnosis was only tive variables. One-way analysis of variance (ANOVA) was
considered once all other possible causes of enteritis/infiltrates used for mean comparison of Ki-67 among groups. P  .05 was
were investigated and excluded.13,14,28,29 A diagnosis of lym- considered statistically significant.
phoma was based on a dense monomorphic population of neo-
plastic lymphocytes with signs of malignancy, such as Results
invasion of the muscularis mucosae, architecture distortion, or
abnormal cell size. The diagnosis of lymphoma-suspect was Clinical Data
based on the presence of a dense focal or patchy monomorphic The age of the dogs in this study was 7.65 + 3.00 years (mean
mucosal infiltrate and/or the presence of intraepithelial lympho- + SD). Twenty dogs (62.5%) were male and 12 (37.5%) were
cytes forming plaques or nests in 1 or more intestinal villi. female. The most represented breeds were Rottweiler (n ¼ 5),
Without prior knowledge of these results, HE sections were Yorkshire terrier (n ¼ 4), boxer (n ¼ 3), cocker spaniel (n ¼ 3),
then studied in conjunction with IHC for CD3 and CD20 by the and gos d’Atura catalán (n ¼ 2).
same pathologists, and a second diagnosis was made. Briefly, Clinical signs at presentation were related to the gastroin-
IBD was diagnosed based on mixed inmunophenotype with testinal tract and included weight loss (96.9%), chronic diar-
CD3þ and CD20þ cells, T-cell lymphoma based on large num- rhea of small bowel origin (81.3%), and chronic vomiting
bers of CD3þ cells with scarce CD20þ cells corresponding to the (53.1%). A remarkable number of cases also had varying
inflammatory background, B-cell lymphoma based on numerous degrees of decreased appetite (53.1%).
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
Case Evaluation
plaques. The diagnoses were revised based on uniformity of cell All 32 cases were diagnosed by histopathology, immunopheno-
labeling, phenotype in association with cell localization, and cell typing, and PARR. Based on morphologic evaluation alone, 7
size as well as growth pattern. The number of cases that were cases were classified as intestinal lymphoma (21.9%), 12 cases
reclassified after immunophenotyping was recorded. Each case as IBD (37.5%), and 13 as lymphoma-suspect (40.6%). When
was analyzed a third time, without knowledge of previous IHC for CD3 and CD20 was analyzed in conjunction with the
diagnosis, by combining histomorphology (HE), IHC, and microscopic findings, 8 cases were classified as T-cell lym-
PARR. The differences in diagnosis at each step were compared. phoma (25%; Fig. 2), 15 cases as IBD (46.9%; Fig. 1), and 9
In addition, morphologic features of crypt distension (normal/ cases as lymphoma-suspect (28.1%). There were no B-cell
mild/moderate/marked), lacteal dilation (normal/mild/moderate/ lymphomas. IHC allowed reclassification of 6 of the 13 cases
marked), villous stunting (normal/mild/moderate/marked), originally classified as lymphoma-suspect; 5 were thought to
mucosal fibrosis (normal/mild/moderate/marked), presence of be IBD and 1 a T-cell lymphoma. However, 2 cases originally
increased intraepithelial lymphocytes (more than 20–30 per diagnosed as IBD were reclassified as lymphoma-suspect
40 stretch of villous epithelium),13 the distribution of the lym- based on the combined HE and IHC results. All 7 cases origi-
phocytic infiltrate (focal, multifocal or diffuse), and monomorph- nally diagnosed as lymphoma remained in the lymphoma group
ism versus polymorphism of lymphocytes based on nuclear size and were further classified as T-cell lymphomas. When PARR
and morphology were recorded for each case. Furthermore, results were used in conjunction with HE and IHC results, all
intraepithelial lymphocytes were assessed in the entire sample 9 cases that had been included in the lymphoma-suspect group
and defined as ‘‘diffuse’’ or ‘‘involving one to several villi.’’34 were diagnosed as IBD, all 15 cases in the IBD group were
The infiltrates of T cells within the epithelium were recorded as diagnosed as IBD, and 8 cases of lymphoma were diagnosed
either surface or crypt infiltrates or both, and intraepithelial infil- as T-cell lymphoma (Tables 1, 2). All lymphomas in this study
trates were divided into single cells, nests, or plaques. Nests of were T-cell lymphomas. The neoplastic lymphocytes were
lymphocytes were defined as 5 clustered intraepithelial classified as large cells in 6 of these cases and as small cell

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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 5

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

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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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 7

Author Contribution proteins (p53, p21 and p16) and heat shock proteins (Hsp27 and
Conception or design: VC, ARB, FRF, AW, RM, TM, MK. Data Hsp70). Vet Pathol. 2011;48:322–329.
acquisition, analysis, or interpretation: VC, ARB, FRF, AW, RM, 11. Coyle K, Steinberg H. Characterization of lymphocytes in canine
TM, MK. Drafting the manuscript: VC, MK. All authors participated gastrointestinal lymphoma. Vet Pathol. 2004;41:141–146.
in critically revising the manuscript, gave final approval, and agree to 12. Dandrieux J, Bornand V, Doherr M, et al. Evaluation of lympho-
be accountable for all aspects of work to ensure integrity and accuracy. cyte apoptosis in dogs with inflammatory bowel disease. Am J Vet
Res. 2008;69:1279–1285.
Acknowledgements 13. Day M, Bilzer T, Mansell J, et al. Histopathological standards
We thank Tom Wood and the DCPAH Histology and Immunohisto- for the diagnosis of gastrointestinal inflammation in endo-
chemistry Laboratory. We also thank the personnel of the DCPAH scopic biopsy samples from the dog and cat: a report from the
Virology Laboratory. We are grateful to the Complutense Veterinary World Small Animal Veterinary Association Gastrointestinal
Teaching Hospital Histology Laboratory for technical support and to Standardization Group. J Comp Pathol. 2008;138(suppl 1):
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Declaration of Conflicting Interests scopic and full-thickness biopsy specimens for diagnosis of
The author(s) declared no potential conflicts of interest with respect to inflammatory bowel disease and alimentary tract lymphoma in
the research, authorship, and/or publication of this article. cats. J Am Vet Med Assoc. 2006;229:1447–1450.
15. Fell J, Walker-Smith J, Spencer J, et al. The distribution of divid-
Funding ing T cells throughout the intestinal wall in inflammatory bowel
Violeta Carrasco’s stay in the DCPH (Michigan State University) was disease (IBD). Clin Exp Immunol. 1996;104:280–285.
financed by the scholarship program of the Spanish Ministry of Educa- 16. Ferrán M. SPSS para Windows. Programación y Análisis Estadı́s-
tion: University Teaching Training Program (Formación de Profesorado tico. Madrid, Spain: McGraw- Hill; 1996.
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