Domestic Animals–Original Article

Veterinary Pathology
2020, Vol. 57(1) 122-131
Chronic Inflammatory and Proliferative ª The Author(s) 2019
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Lesions of the Gallbladder in Aged Pigs DOI: 10.1177/0300985819875749
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Nanako Ushio1, James K. Chambers1 , Ken-ichi Watanabe2,

Takuya E. Kishimoto1, Takanori Shiga1, Jun-You Li3,
Hiroyuki Nakayama1, and Kazuyuki Uchida1

Abstract
Primary epithelial tumors of the gallbladder are rarely reported in animals. In this study, 9 aged pigs (6–12 years old) were
histopathologically examined for gallbladder proliferative lesions. At necropsy, a large gallstone occupied the lumen of the gall-
bladder of 3 pigs. Histopathological examination revealed chronic cholecystitis in all 9 pigs, mucosal hyperplasia in 2 pigs, adenoma
in 1 pig, and adenocarcinoma in 2 pigs. Bacilli were detected in the gallbladder lumen of 6 pigs by Warthin-Starry stain. Mucosal
hyperplasia, adenoma, and adenocarcinoma were characterized by papillary projections of the mucosa with occasional acinar
structures. Tumor invasion of the surrounding tissue was observed in the cases of adenocarcinoma. On Alcian blue and periodic
acid–Schiff double-stained sections, the acinar structure of gallbladder mucosa in chronic cholecystitis and mucosal hyperplasia
was stained in a mosaic pattern, indicating pyloric gland metaplasia. The results of immunohistochemistry revealed a CD10-
positive epithelial brush border and mucin (MUC) 2–positive goblet cells in chronic cholecystitis, adenoma, and adenocarcinomas,
indicating intestinal metaplasia. Immunoreactivity of MUC5 AC and cytokeratin 19 was weaker in adenoma and adenocarcinomas
compared with the normal and hyperplastic gallbladder mucosa. The number of p53-positive nuclei and the Ki-67 index were
higher in adenocarcinomas compared with benign lesions. These results suggest that chronic cholecystitis associated with gall-
stones and/or bacterial infections may contribute to metaplastic changes and development of gallbladder tumors in aged pigs.
Alteration of mucin, cytokeratin, and p53 profiles in gallbladder proliferative lesions in pigs were similar to that in humans,
suggesting a common pathogenesis in tumor development.

Keywords
adenoma, adenocarcinoma, carcinogenesis, cholecystitis, gallbladder, gallstone, hyperplasia, inflammation, progression, swine

Primary epithelial tumors of the gallbladder are rarely reported
in animals. According to a survey of neoplastic diseases in
cattle, gallbladder adenomas are slightly more common in
cows compared with other domestic animals.3,4 Gallbladder
adenocarcinomas have been reported in the dog,6,8,19,28 cat,27
cow3,4 and pig.2,30 In gallbladder adenocarcinomas of the cow
and pig, mucin-secreting columnar epithelial cells were
arranged in a papillary pattern.2–4,30 In gallbladder adenocarci-
nomas of the dog and cat, by contrast, neoplastic cells were
composed of acinar or tubular structures.8,27,28
Gallbladder tumors are also uncommon in humans. 24
Human gallbladder adenocarcinoma is an aggressive disease
with a poor prognosis, and metastases to the lymph nodes and
liver often occur. Prolonged exposure to gallstones can cause
chronic inflammation of the mucosa, which is considered a
major risk factor for developing gallbladder adenocarci-
noma.7,29 Repetitive stimulation to the mucosa and subsequent
tissue repair result in metaplasia and/or dysplasia of the gall-
bladder mucosa, which are considered precancerous lesions
progressing to adenocarcinoma. 14,33,36,37 Sequential
histological and molecular changes over a period of several
years or decades have been studied in human gallbladder dis-
eases.5 Mutation of TP53, a tumor suppressor gene, is com-
monly found in human gallbladder adenocarcinoma, and
accumulation of p53 protein is observed in the tumor cells in
more than 50% of patients. 5,15,17,18,21,38 In addition, the
1
Laboratory of Veterinary Pathology, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Tokyo, Japan
2
Department of Veterinary Medicine, Research Center for Global Agromedi-
cine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro,
Hokkaido, Japan
3
Animal Resource Science Center, Graduate School of Agricultural and Life
Sciences, The University of Tokyo, Ibaraki, Japan
Supplemental material for this article is available online.
Corresponding Author:
Kazuyuki Uchida, Laboratory of Veterinary Pathology, Graduate School of
Agricultural and Life Sciences, the University of Tokyo, 1-1 -1 Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan.
Email: auchidak@mail.ecc.u-tokyo.ac.jp
Ushio et al 123

Table 1. Signalment and Major Pathologic Findings in 9 Pigs With Lesions of the Gallbladder.

Content in Bacteria in Histopathological Diagnosis

Case Breed Age Sex Major Pathological Diagnoses Gallbladder Gallbladder of Gallbladder

1 Yorkshire 7y Male Myocardial degeneration and ND Negative Chronic cholecystitis

fibrosis
Nonsuppurative
meningoencephalitis
2 Yorkshire 9y Female Purulent bronchopneumonia Bile and biliary Negative Chronic cholecystitis
sludge
3 Yorkshire 10y Female Intestinal adenocarcinoma Bile and biliary Negative Chronic cholecystitis
Myelomalacia sludge
4 Yorkshire 7y Male Purulent bronchopneumonia Bile and biliary Bacilli Chronic cholecystitis
Interstitial pneumonia sludge
5 Yorkshire 6y Male Multiple abscesses Gallstone Bacilli Mucosal hyperplasia and chronic
Granulomatous pneumonia cholecystitis
6 Duroc 9y Female Myocardial degeneration and Bile and biliary Bacilli Mucosal hyperplasia and chronic
fibrosis sludge cholecystitis
Ulcerative enteritis
7 Duroc 11y Female Myocardial necrosis Gallstone Bacilli Gallbladder adenoma and chronic
Nonsuppurative cholecystitis
meningoencephalitis
8 Berkshire 12y Female Purulent bronchopneumonia Nothing Bacilli Gallbladder adenocarcinoma and chronic
Gallbladder cholecystitis
adenocarcinoma
9 Duroc 12y Female Purulent bronchopneumonia Gallstone Bacilli Gallbladder adenocarcinoma and chronic
Atherosclerosis of cholecystitis
coronary arteries
Myocardial infarction
Gallbladder
adenocarcinoma

Abbreviation: ND, no data.

production of specific mucins lining the mucosal surface have
been associated with tumor development and progression.1,7,11
The present study examined hyperplastic and neoplastic
lesions in the gallbladder of aged pigs and evaluated their
association with the presence of gallstones and mucosal
inflammation.
Materials and Methods
Cases and Samples
Nine pigs examined in the present report had been kept in the
University Farm of the Graduate School of Agricultural and
Life Sciences, the University of Tokyo. The pigs were raised on
a farm at Fukushima prefecture during the great East Japan
earthquake in March 2011 and had been subsequently kept in
the university farm for observational studies. They were housed
in groups of 3 or 4 in a room with free access to tap water and
were fed a commercial diet (MARUCHIRAKKU; Chubu
Shiryo, Aichi, Japan) at 1.0 to 1.2 kg per head twice a day.
The pigs in this study were emaciated with anorexia and
depression and were euthanized because of poor prognosis
(Table 1). Complete necropsy was performed on all 9 pigs,
which were 6 to 12 years old. At necropsy, visceral organs
including the gallbladder were collected and fixed in 10%
neutral-buffered formalin.
Histopathology
Formalin-fixed tissues were embedded in paraffin. Four-
micrometer-thick sections were stained with hematoxylin and
eosin (HE) for general pathological examinations. Further
examinations for the detection of bacterial agents and mucins
were performed on gallbladder tissue sections with Warthin-
Starry staining and double staining of Alcian blue (AB) and
periodic acid–Schiff (PAS), respectively. For AB-PAS double
staining, deparaffinized sections were first stained with AB (pH
2.5) and subsequently stained with PAS. Tissue sections of the
normal gallbladder, gastric pylorus, and duodenum from a
1-year-old 3-crossbred pig were also stained with AB-PAS.
Immunohistochemistry
Immunohistochemistry (IHC) was performed on gallbladder
tissue sections as described in Table 2, according to the guide-
lines set forth by the American Association of Veterinary Diag-
nosticians Subcommittee on Standardization of
Immunohistochemistry.31 The detection system used in the
present study was the peroxidase-conjugated EnVision
124 Veterinary Pathology 57(1)

Table 2. Primary Antibodies Used for Immunohistochemistry.

Antigen Type (Clone) Antigen retrieval Source

MUC2 Mouse, mAb (Ccp58) Citrate buffer, pH 6.0, microwave Aviva System Biology, San Diego, CA
MUC5AC Mouse, mAb (45M1) Citrate buffer, pH 6.0, autoclave Thermo Fisher Scientific, Fremont, CA
CD10 Mouse, mAb (56C6) Citrate buffer, pH 6.0, autoclave Biogenex, Fremont, CA
CK5/6 Mouse, mAb (D5/16 B4) Target retrieval solution, pH 9.0, autoclave Agilent Technologies, Santa Clara, CA
CK7 Mouse, mAb (OV-TL 12/30) Proteinase K Agilent Technologies, Santa Clara, CA
CK19 Mouse, mAb (b170) Proteinase K Leica Biosystems, Newcastle, UK
CK20 Mouse, mAb (Ks20.8) Proteinase K Agilent Technologies, Santa Clara, CA
Chromogranin A Rabbit, pAb None Yanaihara, Shizuoka, Japan
Ki-67 Mouse, mAb (MIB-1) Citrate buffer, pH 6.0, autoclave Agilent Technologies, Santa Clara, CA
P53 (FL-393) Rabbit, pAb Target retrieval solution, pH 9.0, autoclave Santa Cruz Biotechnology, Santa Cruz, CA
Abbreviations: CK, cytokeratin; mAb, monoclonal antibody; MUC, mucin; pAb, polyclonal antibody.

Polymer Detection System (Agilent Technologies, Santa Clara,
CA). Immunoreactivity of the tumor cells was evaluated for
each marker as follows: –, negative; þ, <25% of tumor cells are
positive; þþ, 25% to 50% of tumor cells are positive; þþþ,
>50% of tumor cells are positive. The number of Ki-67–posi-
tive cells was counted in 1000 epithelial cells on each slide, and
the percentage of Ki-67 positivity (Ki-67 index) was calculated
in each case. The number of p53-nuclear positivity (p53-
positive nucleus number) was counted in 1000 epithelial cells
on each slide, and the percentage of p53-nuclear-positive cells
was calculated in each case.
Results
Gross Findings
At necropsy, dark green friable biliary sludge mixed with bile
filled the gallbladders of 4 pigs (cases 2, 3, 4, and 6). A large
gallstone was observed together with bile in the gallbladder of
3 pigs (cases 5, 7, and 9; Figs. 1 and 2). The gallstones were
yellow to ocher in color and irregular in shape (cases 5, 7, and
9; Figs. 1 and 2). There was no significant lesion in the gall-
bladder mucosa of cases 1 to 4. Gallbladder mucosa was
slightly thickened in cases 5 and 6. In case 7, a focal nodule
was located in the fundus of the gallbladder. In case 9, the
gallbladder wall was diffusely and irregularly thickened, and
multifocal hemorrhage was observed on the surface of the
thickened mucosa (Fig. 3). The liver was yellowish, and the
intrahepatic bile ducts were severely dilated with bile, indicat-
ing the biliary tract obstruction. In case 8, the lumen contained
no bile fluid or gallstones but was replaced by the proliferation
of soft white neoplastic tissues (Fig. 4).
Histopathology
Diffuse infiltration of lymphocytes and plasma cells as well as
hyperplastic lymphoid follicles were observed in the gallblad-
der mucosa of all 9 pigs (Fig. 5). Warthin-Starry staining
revealed bacilli in the gallbladder lumen of 6 pigs (cases 4–
9) and in the gallstone of 1 pig (case 8). Diffuse or focal
hyperplasia of the gallbladder mucosa with acinar structures
and elongated folds was observed in 2 pigs (cases 5 and 6;
Fig. 6). The acinar cells of the mucosa in chronic cholecystitis
and mucosal hyperplasia had clear mucinous cytoplasm with an
eccentric nucleus (Fig. 6, arrow). In contrast, surface epithelial
cells composing the folds had the eosinophilic cytoplasm, and
goblet cell-like cells were occasionally observed.
Neoplastic lesions of the gallbladder in 3 pigs included ade-
noma (case 7) and adenocarcinoma (cases 8 and 9). Adenoma
was composed of tubular and papillary structures with thin
stroma lined by a single layer of neoplastic epithelial cells (Fig.
7). Most of the neoplastic epithelial cells had eosinophilic cyto-
plasm, occasionally with a brush border on the cell membrane.
Goblet cell-like cells were also occasionally observed. Aniso-
karyosis was mild, and nuclei were located in the basal side of
the cell. In the pig with gallbladder adenoma (case 7), the
extrahepatic duct was obstructed by a gallstone, and the
mucosa was necrotic.
Adenocarcinomas were composed of papillary structures
lined by single to multiple layers of epithelial cells that invaded
the muscularis of the gallbladder (Figs. 8 and 9). In case 9, the
tumor cells further invaded the liver and the gastrohepatic
lymph node. Severe anisokaryosis and loss of cellular and
nuclear polarity of the neoplastic cells were evident in adeno-
carcinoma. Squamous metaplasia with keratinization was occa-
sionally observed in the invasive lesion of adenocarcinoma
(Fig. 10, arrows), and basal cell-like tumor cells were arranged
in solid sheet structures (Fig. 11).
Mucins and Immunohistochemical Profiles
Results of AB-PAS stain and IHC for normal tissues and
inflammatory and proliferative gallbladder lesions are summar-
ized in Table 3.
On AB-PAS double-stained sections, acid mucin is stained
blue with AB, neutral mucin stains magenta with PAS, and a
mixture of both stains purple. Surface epithelial cells and
glandular acinar cells of the normal gallbladder mucosa were
stained purple in the luminal surface and in the cytoplasm
(Suppl. Fig. S1). In the normal pyloric mucosa, surface epithe-
lial cells were stained magenta (Suppl. Fig. S2), while cells of
the pyloric gland were stained in a mosaic pattern of magenta
and blue (Suppl. Fig. S3). In the normal duodenal mucosa, the
Ushio et al 125

Figures 1–4. Figure 1. Adenoma, gallbladder, pig, case 7. The gallbladder contains a large gallstone and bile. Figures 2–3. Adenocarcinoma,
gallbladder and liver, pig, case 9. The enlarged bile duct and gallbladder are filled with yellow gallstones. The liver is discolored yellow, and the
common hepatic duct is enlarged and filled with yellow gelatinous material. The gallbladder wall is irregularly thickened and convoluted, and
there is multifocal hemorrhage. Figure 4. Adenocarcinoma, gallbladder, pig, case 8. The gallbladder is shrunken (arrows) and contains no bile.
The gallbladder wall is severely thickened, and the lumen is occupied by white multinodular tissue.

brush border of the surface epithelial cells was stained
magenta, and the cytoplasm of the goblet cells was stained
purple (Suppl. Figs. S4 and S5).
Most surface epithelial cells in chronic cholecystitis, muco-
sal hyperplasia, and adenoma were stained purple on the lumi-
nal surface and diffusely purple in the cytoplasm (Fig. 12),
similar to the staining pattern of the normal gallbladder
mucosa. In some areas of chronic cholecystitis and adenoma,
goblet cell-like cells with purple cytoplasm and magenta brush
border were observed (Fig. 13), which resembled the appear-
ance of the duodenal mucosa. Most of the acinar cells of
chronic cholecystitis, mucosal hyperplasia, and adenoma were
stained purple on the luminal surface and in the cytoplasm. In
some areas of chronic cholecystitis and mucosal hyperplasia, a
mosaic pattern of magenta- and blue-stained acinar cells was
observed (Fig. 14), which resembled the appearance of the
pyloric gland. The cytoplasm of adenocarcinoma cells was
negative for both AB and PAS.
The results of IHC revealed that surface epithelial cells and
glandular acinar cells of the normal gallbladder mucosa were
positive for mucin (MUC) 5 AC, cytokeratin (CK) 7, CK19,
and CK20 (Suppl. Figs. S6, S7, and S8) and negative for
MUC2, CD10, and CK5/6. Few chromogranin A–positive neu-
roendocrine cells were randomly observed in the gallbladder
mucosa (Suppl. Fig. S9). Surface epithelial cells of the normal
pyloric mucosa were positive for MUC5 AC (Suppl. Fig S10)
and negative for MUC2 and CD10, while the epithelial cells of
pyloric glands were negative for MUC5 AC, MUC2, and
CD10. In the normal duodenal mucosa, the goblet cells were
positive for MUC2 (Suppl. Fig. S11) and negative for MUC5
AC and CD10, and the brush border was positive for CD10
(Suppl. Fig. S12).
Surface epithelial cells and glandular acinar cells in chronic
cholecystitis, mucosal hyperplasia, and adenoma were positive
for MUC5 AC (Fig. 15), CK7, and CK19 but negative for
CK5/6. Surface epithelial cells and glandular cells were also
positive for CK20 in chronic cholecystitis and adenoma but
negative in mucosal hyperplasia. The acinar cells that appeared
in a mosaic pattern of blue and magenta by AB-PAS staining
were negative for MUC5 AC in chronic cholecystitis and
126 Veterinary Pathology 57(1)

Figures 5–11. Figure 5. Chronic cholecystitis, gallbladder, pig, case 4. A diffuse infiltrate of lymphocytes and plasma cells and a lymphoid follicle
are present in the gallbladder mucosa. Hematoxylin and eosin (HE). Figure 6. Hyperplastic cholecystitis, gallbladder, pig, case 5. Diffuse
hyperplasia and lymphocytic inflammation of the gallbladder mucosa. The gallbladder mucosa is thickened with acinar structures (arrows) and
elongated mucosal folds. HE. Figure 7. Adenoma, gallbladder, pig, case 7. A pedunculated polyp is composed of neoplastic epithelial cells
arranged in tubular and papillary structures with fibrous stroma. HE. Figure 8. Adenocarcinoma, gallbladder, pig, case 8. Atypical epithelial cells
are arranged in papillary structures. HE. Figures 9–11. Adenocarcinoma, gallbladder, pig, case 9. Figure 9. Atypical epithelial cells are arranged
into papillary structures supported by a thin fibrous stroma, and neoplastic basaloid cells form solid sheets. Neoplastic epithelial cells infiltrating
the gallbladder muscularis (lower left) exhibit squamous metaplasia. HE. Figure 10. In areas of squamous metaplasia, there is keratinization of
neoplastic epithelial cells (arrows). HE. Figure 11. Neoplastic epithelial cells arranged into solid sheets have round or oval basophilic nuclei with
scant eosinophilic cytoplasm. HE.
Ushio et al 127

Table 3. Mucin and Immunohistochemical Profiles of Epithelial Cells in Control Tissues and the Gallbladder Lesions.

AB-PAS
Staining Ki-67 p53-Positive
Pattern MUC5ACa MUC2a CD10a CK5/6a CK7a CK19a CK20a Cg Aa Index (%)b Nuclei, %c

Control tissues
Normal gallbladder
Surface epithelium Purple þþþ – – – þþþ þþþ þþ þ 5.3 0.3
Mucous gland Purple þþþ – – – þþþ þþþ þþ þ
Pylorus
Surface epithelium Magenta þ – – – – þ – – NT NT
Pyloric gland Blue- – – – – þ – þ þ
magenta
Duodenum
Surface epithelium Magenta – – þ – – þ – þ NT NT
Goblet cell Purple – þ – – – þ – –
Gallbladder lesions
Chronic cholecystitis
Case 1 Purple þþþ – – – þþþ þþþ þþ þ 5.5 0.3
Case 2 Purple/ þþþ þ þ – þþþ þþþ þþ þ 5.1 0.4
blue-
magenta
Case 3 Purple/ þþþ þ þ – þþþ þþþ þþ þ 4.8 0.9
blue-
magenta
Case 4 Purple/ þþþ þ þ – þþþ þþþ þ þ 6.3 1.6
blue-
magenta
Mucosal hyperplasia
Case 5 Purple/ þþþ – – – þþ þþþ – – 8.0 3.5
blue-
magenta
Case 6 Purple þþþ – – – þþ þþþ – – 17.6 21.5
Adenoma
Case 7 Purple þþ þ þ – þþþ þþ þþ – 36.6 24.4
Adenocarcinoma
Case 8 – þ – – þ þþ þþ þ þ 82.6 55.3
Case 9 – þ þ þ þ þþ þþ þ þ 94.6 96.5
Abbreviations: AB-PAS, double staining of Alcian blue and periodic acid–Schiff; MUC, mucin; CK, cytokeratin; Cg A, chromogranin A; NT, not tested.
a
Immunohistochemical scoring: –, negative; þ, 1%–25% positive cells; þþ, 26%–50% positive cells; þþþ, >50% positive cells.
b
Percentage of Ki-67 positivity (Ki-67 index) was calculated based on the number of Ki-67-positive cells counted in 1000 epithelial cells.
c
Percentage of p53 positivity (p53-positive nucleus number) was calculated based on the number of p53-nuclear positive cells counted in 1000 epithelial cells.

mucosal hyperplasia. In chronic cholecystitis and adenoma, the
brush border of mucosal epithelial cells was positive for CD10
(Fig. 16), and the cytoplasm of goblet cell-like cells were pos-
itive for MUC2 (Fig. 17). In the lesions of adenocarcinoma, the
mucosal epithelial cells were positive for CK7, CK19, CK20,
and chromogranin A (Suppl. Figs. S13–S15). Basal cell-like
neoplastic cells and the invasive cells in adenocarcinoma were
positive for CK5/6 (Fig. 18). In some cells, cytoplasmic mucin
was positive for MUC5 AC and MUC2. The brush border in the
adenocarcinoma lesions was positive for CD10.
Ki-67 indices were 5.3% in the normal gallbladder mucosa;
5.5% (case 1), 5.1% (case 2), 4.8% (case 3), and 6.3% (case 4)
in chronic cholecystitis (mean 5.4%); 8.0% (case 5) and 17.6%
(case 6) in mucosal hyperplasia (mean 12.8%); 36.6% (case 7)
in adenoma; and 82.6% (case 8) and 94.6% (case 9) in adeno-
carcinomas (mean 88.7%; Figs. 19–22).
The normal gallbladder mucosa was negative for p53. Few
p53-positive cells were observed in a hyperplastic gallbladder
(case 6) and adenoma (case 7; Figs. 23 and 24). A large number
of p53-positive cells were observed in adenocarcinomas, espe-
cially in basal cell-like cells and invasive cells (cases 8 and 9;
Fig. 25).
Discussion
The present study describes histopathological findings of gall-
bladder epithelial tumors in 3 aged pigs: 1 adenoma and 2
adenocarcinomas. Also, gallbladder mucosal hyperplasia was
observed in another 2 pigs. Chronic inflammation and meta-
plastic changes were also observed within these proliferative
lesions and in the other pigs investigated. Although the pigs in
the present study once lived in the evacuation zone in
128 Veterinary Pathology 57(1)

Figures 12–18. Figure 12. Mucosal hyperplasia, gallbladder, pig, case 5. Surface epithelial cells are stained purple at the luminal surface and
diffusely purple in the cytoplasm. Alcian blue with periodic acid–Schiff (AB-PAS). Figure 13. Adenoma, gallbladder, pig, case 7. The brush border
of neoplastic epithelial cells is stained magenta, and the cytoplasm of goblet like cells is stained purple. AB-PAS. Figure 14. Mucosal hyperplasia,
gallbladder, pig, case 5. Acinar structures are stained in a mosaic pattern of magenta and blue. AB-PAS. Figure 15. Mucosal hyperplasia,
gallbladder, pig, case 6. Luminal surface of the mucosal epithelial cells and the cytoplasm of goblet cell-like cells are positive for mucin (MUC) 5
AC. Immunohistochemistry (IHC) for MUC5 AC. Figure 16. Adenoma, gallbladder, pig, case 7. Brush border of the neoplastic epithelial cells is
positive for CD10. Inset: high magnification. IHC for CD10. Figure 17. Adenoma, gallbladder, pig, case 7. Cytoplasmic mucin in some neoplastic
epithelial cells and goblet cell-like cells are positive for MUC2. Inset: high magnification. IHC for MUC2. Figure 18. Adenocarcinoma,
gallbladder, pig, case 9. Cytokeratin labeling in neoplastic epithelial cells arranged in solids sheet (comprising basaloid cells, arrow) and an
invasive lesion (arrowhead). IHC for CK5/6.

Fukushima Prefecture and might have been exposed to radio-
active pollutants, we could not conclude the impact of radiation
exposure on gallbladder carcinomas. Recently, pathological
research of Japanese black cattle living in the same evacuation
zone revealed that there was no significant relationship
between the radiation effect and incidence of tumors.32 Thus,
we expect that radiation exposure is not likely related to the
tumorigenesis of the gallbladder carcinoma in pigs.
Gallstones are classified into 3 types according to the
amount of cholesterol and calcium bilirubinate: cholesterol
gallstones, pigment gallstones, and mixed gallstones.35 The
gross color of cholesterol gallstones is yellow and that of pig-
ment gallstones is brownish or black.9 Cholesterol gallstones
are formed and enlarged by biliary cholesterol supersaturation
due to metabolic alterations and hypersecretion of mucins,12
which can be induced by the intake of a cholesterol-enriched
diet also in pigs.10,23 In a study on spontaneous lesions in
Göttingen minipigs, cholecystitis was observed especially in
pigs older than 6 months.20,22 The gallstones in the present
pigs, based on gross inspection, are considered to be mainly
composed of cholesterol, although the pigs were not fed
cholesterol-enriched foods. The formation of cholesterol gall-
stones in aged pigs might be promoted by 2 factors: hepatic
cholesterol synthesis and chronic cholecystitis-induced mucin
hypersecretion.
The pathogenesis of gallbladder adenocarcinoma has been
studied mainly in tissues from human patients, and 2 pathways
are proposed. One is the adenoma-carcinoma cascade, which is
considered a minor pathway because gallbladder adenomas are
comparatively rare.1 The other pathway is sequential steps of
chronic inflammation, metaplasia, dysplasia, and finally ade-
nocarcinoma.37 This multistep pathogenesis is considered more
plausible because chronic inflammation, hyperplasia, and
metaplastic changes are often seen in the vicinity of
Ushio et al 129

Figures 19–22. Gallbladder, pig. Immunohistochemistry (IHC) for Ki-67. Figure 19. Normal mucosa, 1-year-old pig. Nuclear expression of Ki-
67 in few mucosal epithelial cells. Figure 20. Mucosal hyperplasia, case 6. Nuclear expression of Ki-67 is present in increased numbers of
epithelial cells. Figure 21. Adenoma, gallbladder, pig, case 7. Nuclear expression of Ki-67 is detected in frequent epithelial cells. Figure 22.
Adenocarcinoma, gallbladder, pig, case 9. Nuclear expression of Ki-67 in most epithelial cells. Figures 23–25. Gallbladder, pig. IHC for p53.
Figure 23. Mucosal hyperplasia, case 6. Nuclear expression of p53 in few mucosal epithelial cells. Inset: high magnification. Figure 24.
Adenoma, case 7. Nuclear expression of p53 in few neoplastic epithelial cells. Inset: high magnification. Figure 25. Adenocarcinoma, case 9.
Nuclear expression of p53 in most neoplastic cells.

adenocarcinoma in humans. Chronic inflammation commonly
results from gallstones, changes in the bile nature, and bacterial
infection.14 In the present study, bacilli were detected in the
gallbladder of 6 pigs, and 5 of those 6 pigs developed gallblad-
der mucosal hyperplasia (cases 5 and 6), adenoma (case 7), and
adenocarcinoma (cases 8 and 9). Gallstone was detected in 3
pigs affected with gallbladder mucosal hyperplasia (case 5),
adenoma (case 7), and adenocarcinoma (case 9). In 5 pigs,
mucosal epithelial cells with CD10- and PAS-positive brush
border and MUC2-positive goblet cells were observed, indicat-
ing intestinal metaplasia of the gallbladder mucosa. Two of the
5 pigs developed adenoma (case 7) and adenocarcinoma (case
9). In 4 pigs, a mosaic pattern of AB-PAS–stained acinar cells
was observed in the gallbladder mucosa, indicating pyloric
gland metaplasia. One of the 4 pigs developed mucosal hyper-
plasia (case 5).
Phenotypic alterations of mucins and cytokeratins have been
studied in human gallbladder tumors.11,33 The expression level
of MUC5 AC is reduced in adenoma and adenocarcinoma,
compared with the normal gallbladder mucosa that diffusely
expresses MUC5 AC. In the present study, MUC5 AC was
diffusely expressed in the normal, inflammatory, and hyper-
plastic gallbladder of the pig. Similar to the human gallbladder
tumor, the expression of MUC5 AC was reduced in adenoma
and adenocarcinoma of the pig. With regard to the cytokeratin
profiles of tumor cells, loss of CK7 expression was associated
with a poor survival of gallbladder adenocarcinoma patients.11
Also, CK20 expression was associated with carcinomatous
changes of gallbladder adenoma in human patients. In the pres-
ent study on the pig gallbladder, CK7 and CK19 were diffusely
and consistently expressed in the normal mucosa, chronic cho-
lecystitis, mucosal hyperplasia, adenoma, and adenocarcinoma.
CK20 was expressed also in the normal gallbladder, chronic
cholecystitis, adenoma, and adenocarcinoma but absent in the
hyperplastic gallbladder mucosa.
Chromogranin A expression was observed in adenocarcinoma
lesions of 2 pigs (cases 8 and 9), suggesting the multipotent nature
of the tumor cells. In humans, chromogranin A–positive adeno-
neuroendocrine carcinomas of the gallbladder have been
reported, and multipotent stem cells in the gallbladder mucosa
130
or neuroendocrine cells in the intestinal or gastric metaplasia
lesions have been considered as the origin of the tumor.13,26 In
the present study, intestinal metaplasia was observed in 1 of the
pigs with gallbladder adenocarcinoma (case 9).
Aberration of a tumor-suppressor gene, TP53, is frequently
detected in human gallbladder adenocarcinomas (more than
90%). Moreover, TP53 abnormality has been detected in
57% of the gallbladder tissue from human gallstone patients
with chronic cholecystitis and without adenocarcinoma; thus, it
is considered to be one of the earliest changes during gallblad-
der tumor development.25 Missense mutation of TP53 corre-
lates with the higher level of p53 nuclear expressions in
humans.38 Moreover, the nuclear expression of p53 correlates
with poor differentiation of tumor cells, tumor size, lymph
node metastasis, and high invasiveness in human gallbladder
adenocarcinoma.16,34 In the present study, p53 expression was
absent in the normal gallbladder of the pig. Focal expression of
p53 was detected in pigs with hyperplastic lesions of the gall-
bladder (case 6) and also with gallbladder adenoma (case 7).
Intense p53 expression was observed in gallbladder adenocar-
cinomas (cases 8 and 9). These results indicate that changes in
the p53 expression may be associated with gallbladder carci-
nogenesis in the pig, as well as in humans. Also, p53 nuclear
expression was highly detected in the invasive and solid lesions
compared with the papillary lesions of adenocarcinomas, indi-
cating that the p53 nuclear expression may be correlated with
differentiation and invasiveness of gallbladder adenocarcinoma
in the pig, as well as in humans.
In conclusion, persistent chronic cholecystitis associated
with gallstones and bacterial infections may trigger the onset
of hyperplasia and adenoma with metaplasia and ultimately
develop into adenocarcinoma in the pig gallbladder, as well
as in humans. It is likely that aged pigs develop gallbladder
adenocarcinoma because this multistep carcinogenesis pro-
gresses over decades.
Acknowledgements
We thank Ms Shizuka Kato for her technical assistance.
Declaration of Conflicting Interests
The authors declare no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The authors received no financial support for the research, authorship,
and/or publication of this article.
ORCID iD
James K. Chambers https://orcid.org/0000-0001-5273-7221
Kazuyuki Uchida https://orcid.org/0000-0002-2302-799X
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