Histopathology
of Urinary Bladder Tumors
Induced
Bovine Cutaneous
Papilloma Agent1
by
D. F. BROBST2AND C. OLSON
(Department
of Veterinary
Science,
University
of Wisconsin,
Madison,
Wisconsin)
SUM@.{ARY
Fibromatous tumors were induced in the wall of the urinary bladder of calves by
injecting suspensions of bovine cutaneous papillomas into the submucosa. The
urinary
bladder
tumors were examined
histologically
after 15-500 days of growth to
determine the sequence and degree of change. The fibromas were invasive but did
not metastasize. Various lesions also occurred in the bladder mucosa, apparently in
association with the induced fibromas. These included polypoid hyperplasia of the
mucosa and squamous and glandular metaplasia of the transitional epithelium. Pre
vious exposure of calves to wart vaccine modified the histologic pattern of the bladder
lesions slightly. These tumors, when present, were generally smaller than in non
vaccinated calves. In some tumors the polypoid projections of mucosa regressed,
although fibromatous tissue was present at the site of injection in the bladder wall. In
three animals polypoid projections
of mucosa were present 1038 days after inoculation.
The ability of the bovine cutaneous papifioma agent
to induce tumors
in the bovine urinary
bladder
as well
as in the skin and vagina was previously reported (7, 8).
The induced tumors were benign in histologic appearance
and no metastases were observed.
This
paper
examined
at
reports
the
intervals
histology
varying
of bladder
from
15-500
tumors
days
after
induction with the papilloma agent.
MATERIALS AND METHODS
Five different cutaneous papifioma isolates (215, 219,
233-A, 247, and 260) were used for inoculation of the skin
and urinary bladder in 59 nonvaccinated and twenty
vaccinated calves 2—6mo. of age. Some of the non
vaccinated and all of the vaccinated calves were part of a
previously reported experiment on immunity (7). Inocula
were prepared as a 10 % isotonic saline suspension of bovine
wart tissue which had been stored in 50 per cent glycerine
at 4°C. Duplicate injections of approximately 0.5 ml of
inoculum were made into the submucosa of the urinary
bladder either by suprapubic cystostomy or, in female
calves, transurethrally with a cystoscope and a No. 28
Stem-McCarthy working element. The inoculum used
in the urinary
1 Published
Agricultural
bladder
with
was also inoculated
approval
Experiment
of RG, C4627 National
Research
Committee
Wisconsin
Alumni
2 Present
Purdue
address:
University,
of
Station.
the
Director
Supported
Institutes
of the Graduate
Research
School
Lafayette,
of
the
in part
Wisconsin
with the aid
and by the
School with funds from
Foundation.
Indiana.
Received for publication July 11, 1964.
Science
study
tumor
growth
and
Medicine,
and regression.
Bladder
lesions in
35 nonvaccinated calves were examined histologically
15—500days after inoculation.
Twenty calves were vaccinated with formalinized
cutaneous papilloma suspensions to test the ability to
immunize of wart isOlates 215 and 260. Ten calves re
ceived vaccine prepared from material 215 and ten re
ceived vaccine prepared from material 260. The vaccines
were prepared by making a 5 % suspension of ground wart
material and adding formalin to make a final concentration
of 0.5 %. The vaccination procedure consisted of 0.5
ml vaccine
intradermally
which was repeated
ten days
later. Twenty days after completion of vaccination the
urinary bladder, skin, and other tissues were challenged
with inocula prepared from materials 215 and 260. The
methods
of inoculation
were as in the unvaccinated
calves.
Five of each group of ten calves vaccinated with materials
215 and 260 were challenged with material 215 and five
with material 260. All animals were examined cystoscopi
cally at monthly intervals and all vaccinated calves were
killed 117—150
days from the time of challenge.
intradermally
of Health,
of Veterinary
and on scarified skin of the same animal. Female calves
were examined cystoscopically at monthly intervals to
RESULTS
Nonvaccinated
calves.—The five
cutaneous
papilloma
isolates used in inoculation were active and produced
growths
in either skin or urinary
247, did not produce
bladder
lesions
bladder.
One isolate,
but did cause cutane
ous papillomas. Forty-eight of the 59 calves developed
bladder tumors which were detected cystoscopically,
and 38 grew warts at the skin injection sites (Table 1).
12
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
BROBST AND OLSON—Bladder Tumors and a Papilloma
TABLE 1
Agent
13
killed, the number with tumor at the beginningof each observa
The dense tissue in the base of all lesions consisted of a
compact mass of fibroblasts. From 60 to 180 days after
inoculation this fibromatous tissue began to infiltrate the
edematous tissue of the polyps (Fig. 3). Although the
fibromatous reaction was located primarily within the
lamina propria and submucosa, fibroblasts were observed
tion period
penetrating
COMPARISON OF REGRESSION OF 48 BLADDER TUMORS AND 38
SKIN TUMORS PRODUCED BY INOCULATION OF CUTANEOUS
PAPILLOMA MATERIALS IN 59 NONYACCINATED CALVES
Since some tumors regressed and other tumor-bearing calves were
is smaller
than in the preceding
period.
———
TumorsiteObservation
@
791—900Bladder― 0-60
Skin1/48b
a
Results
are
61—120 121—180181—300301—500
3/381/41 9/301/265/120/112/51/10 1/2@1/60/3°
based
b Numerator
=
on
Number
cystoscopic
of
tissue
in
which
the
tumor
observation period.
calves
had
small
lesions
which
were
observed
cysto
scopically 1038 days after inoculation.
‘No
skin
growth
lasted
longer
than
362
days.
Thirty-seven calves in which tumors were observed cys
toscopically were killed. The bladder tumors in 35 of
these calves were examined at necropsy 15-500 days after
inoculation.
The majority of the bladder lesions were composed of
polypoid projections of mucosa arranged about the in
jection sites (Fig. 1). In some lesions the polypoid pro
jections were located about the rim of a crater void of
mucosal projections (Fig. 2). The polyps of each tumor
were of various sizes but in the majority
of the lesions the
polyps did not exceed 1.5 cm in length and 5 mm in diame
tsr. The maximum size of mucosal projections was found
in a tumor 374 days old which had projections measuring
1-4 cm in length and 3—10mm in diameter.
deep in the bladder
epithelium
(Fig. 5).
of lesions less than 180 days old had polyps which were
pliable and slightly translucent, whereas in tumors older
than 180 days the polyps were more rigid and opaque.
The base of the lesions in cross section consisted of dense
tissue and often was sharply delineated from normal
bladder wall (Fig. 2).
areas
observed in tumors
cells, many
of which were goblet-shaped
and filled with
material that stained like mucin. Columnar cells oc
casionally were present within buds of transitional epi
thelium (Fig. 7), but more commonly were present lining
crypts of mucosa. In cross section these crypts had the
of glands (Figs. 8, 9).
Squamous
metaplasia
was observed less commonly in the epitheium of the
polyps than was glandular metaplasia. It was observed
most often in epithelium which was in direct contact with
fibromatous
tissue that had infiltrated
the polyps and in
epithelium lining a central crater present at the base of
some lesions. The stroma of the polypoid projections of
tumors less than 180 days old was largely lamina propria;
TABLE 2
SUMMARY OF HISTOPATROLOGIC FINDINGS IN BLkDDER TUMORS INDUCED IN 35
NONVACCINATED
AND TEN
VACCINATED
CALVES
@HANOES
(PERcENTINVOLVEMENT)
No. or CALVESDURATION
(DAYS)Eprrxzuaz.
Polyp
forina.
tion
laxityNonvaccinated
5
14
61—120
13
121—180
3
181—500
100
100
100
Thickened
epithe-
Intraepitheial
Glandular
mets-
Squa
inous
mets.-
93
71
70
14
23
7
15
60
66
33
hum
85
66
CHANGES
INVOLVEMENT).
(PERCENT
Fibro
blut
prolifer
cystsEd@1flaIn
cysts plasia plulaSuus@pii@siau@i@
ation
@@jj5j
100
100
100
43
15
66
100
61
54
100
66
100
Vaccinated
1015—60
117—15080
of
cysts were
older than 60 days (Table 2). Subepithelial cysts were
present within the polyps of many tumors and were most
common in growths more than 180 days old. These
cysts were observed within nests of transitional epithelium
(Brunn's nests) apparently isolated from the surface
(Fig. 6), or in the process of becoming isolated. These
cysts frequently contained material which stained red
with mucicarmine stain. Glandular and squamous meta
plasia were observed in the epithelia of the polyps of many
tumors. Glandular metaplasia was observed only in
polyps of tumors more than 60 days old and was seen in
66 % of the tumors more than 180 days old (Table 2).
The glandular epithelium was composed of columnar
appearance
The majority
Within
small intraepithelial
often present and were most frequently
re
greased. Denominator = Number of animals with tumor in each
C These
muscle bundles
were in juxtaposition
thickened
observations.
animals
between
wall. In a lesion of 500 days duration, fibroblastic tissue
was present within the polyps and extended to the serosal
surface of the bladder (Fig. 4).
The histology of the polypoid projections of mucosa
varied with their age. In general the epithelium was
thicker than normal and often the basal layer of cells was
indistinct where transitional epithelium and fibromatous
period (days)
8080 8020 600
2020 80100 10020 2010010040 30
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
Vol. 25, January 1965
Cancer Research
14
evidence of chronic inflammation, as represented by
edema and inifitration with round cells, was present.
In tumors older than 180 days the stroma of the polypoid
only of fibromatous masses in the submucosa which were
projections
like lesions with a firm fibrous base surrounded
was
largely
of fibromatous
tissue
with
mature
collagen often present. In some projections which had
been infiltrated with fibromatous tissue there was an
increase
increase
junction
in the number and size of blood vessels. This
in vascularity was located most often near the
of fibromatous
reaction and transitional
not apparent
on gross examination.
The majority
of the
remaining growths in the vaccinated group were crater
bulbous polyps.
by short
Microscopically the epithelial and sub
epithelial alterations
were similar to those observed in
tumors seen in the nonvaccinated
control animals (Table
2). Squamous metapla.sia of the epithelium was a more
common finding in bladder lesions of vaccinated than in
epi
nonvaccinated
animals.
This metaplastic alteration was
observed primarily in the central part of the lesion which
thelium (Fig. 10).
The polypoid projections of the tumors in five calves
regressed to the point where they were no longer apparent
was not covered with polyps.
by cystoscopy (Table 1). Regression was a gradual proc
ess in which the projections first lost their pink edematous
appearance and became firm and white. At subsequent
examinations the polyps became shorter, more blunt, and
the base of the crater
Areas of ulceration within
were rarely
observed.
DISCUSSION
finally assumed an appearance resembling normal bladder
mucosa.
Cystoscopic examination of a bladder tumor at
The most constant feature of the tumor induced in the
urinary bladder of cattle was a fibromatous mass which
developed at the site of submucosal injection. This tissue
500 days revealed the polyps to be much shorter than
resembled
previously noted and, at necropsy, a thickened fibrous
mass was still present in the bladder wall (Fig. 4). In
bovine cutaneous papilloma, and vaginal tumors of cattle
induced with the bovine cutaneous papilloma agent (1,
5). Mitotic figures were rarely seen in the fibromatous
three animals, short firm polypoid projections of mucosa
were observed cystoscopically 1038 days after inoculation.
The cutaneous papillomas of twenty calves regressed
spontaneously
35—362days after inoculation
(Table 1).
But regression of cutaneous papillomas did not coincide
with regression
of the polyps of the bladder
lesions.
Vaccinated calves.—Three of the ten calves given in
jections of vaccine prepared
from material
the fibromatous
element,
although
it did exhibit
seen in the base
a definite
of the
invasiveness.
In tumors with a duration of less than 60 days the fibro
blastic tissue was generally confined to the area of the
injection site. In older tumors the fibroma extended into
the edematous polyps, and in a tumor of 500 days duration
the entire anterior third of the bladder was composed of
dense fibromatous
260, and seven
tissue
tissue.
There was no evidence to
of the ten calves receiving vaccine prepared from material
indicate
215,
nant, since the tissue appeared to become less cellular and
more mature with age.
The projections of mucosa formed by elevations of the
developed
bladder
growths developing in
calves varied in size and
smaller than growths of
cinated control calves.
tumors
when
challenged.
The
the urinary bladders of these
appearance and all but one were
similar duration in the nonvac
Two of the tumors consisted
FIG.
1 .—Mucosal
aspect
that the fibromatous
edematous
lamina propria
tissue would become malig
in some respects resembled
of the
urinary
bladder
of a nonvac
cinated calf with papillary and polypoid projections at the in
jection site. The bladder was inoculated with bovine papilloma
material 123 days prior to necropsy.
FIG.
2.—Cross
section
through
urinary
bladder
tumor
of non
vaccinated calf which was inoculated with bovine papilloma ma
terial 191 days prior to necropsy.
Arrows indicate the thickness
of fibromatous tissue at site of injection.
FIG. 3.—Section
nonvaccinated
calf
loma material.
through
79 days
base
after
Fibromatous
FIG.
4.—Cross
section
of polyps
of bladder
tumor
in a
its induction
with bovine
papil
tissue in base of the lesion has in
filtrated a projection of mucosa.
through
X 50.
urinary
bladder
tumor
in non
vaccinated calf which was inoculated with bovine papilloma ma
terial 500 days prior to necropsy. Arrows indicate the thickness
of fibromatous tissue in wall of the bladder.
FIG.
5.—In
nonvaccinated
calf,
section
through
two
poiyps
of
bladder tumor induced with bovine papilloma material 316 days
prior
to necropsy.
Polyp
in the
upper
right
of photomicrograph
was edematous, and the basal layer of the epithelium was distinct
in contrast to that present in the polyp shown in lower part of
the photomicrograph.
Numerous plasma cells and lymphocytes
were present in the irregular j unctional area of epithelium and
fibromatous
FIG.
element.
X 100.
6.—Photomicrograph
the
papillae seen in naturally occurring papillary hyperplasia
of the
tumor
illustrated
in Fig.
4.
This subepithelial cyst was present within a polypoid projection
of the tumor. Mature fibrous tissue similar to that in base of
the lesion is on the right of photomicrograph.
X 100.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
@
•
@_
:@ t:4@_;v
3
_j.,,ll._
I@@@[email protected]
I
..@ ‘)..
-
@
.
@
h@ETRtC'SYST(M@
@
.
@
@
k,..'
@
@
@
-.@
@:
3@
3
I'
. .
@I
..
‘I,?
@...
.@__r
,@
,@
@k@'%,i:
..
t
Pg.'
1,_I
.‘..,.
. .
‘
.I@
.
.@
.
•
‘I@t
@‘...
r
-I.
o—
1@.
.
@.%
15
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
FIG.
7.—Section
through
a polyp
of the
tumor
also
illustrated
by the photomicrograph
in Fig. 5. The proliferation of the epi
thelium was toward the lamina propria and appeared to be in
process of becoming isolated from the surface epithelium.
Arrow
points to gland-like arrangement
of columnar cells within the
proliferating bud of cells. X 400.
FIG. 8.—Section
through
a portion
of tumor
described
in Fig.
5.
A large portion of the epithelium contained metaplastic columnar
cells.
The
lamina
propria.
metaplastic
epithelium
At the bottom
has
proliferated
toward
of photomicrograph
there
the
is dense
fibromatous
tissue.
X 30.
FIG. 9.—A higher magnification
of a different area of the tumor
illustrated
in Figs. 5, 7 and 8, depicting
the changes of cystiti8
glandularis.
The nests of metaplastic
of columnar
and goblet-shaped
FIG. 10.—Hemangiomatous
vaccinated
days prior
were
to necropsy.
FIG.
where
tissue
Numerous
thickened
of tumor
11.—Submucous
were composed
bladder
with papilloma
thin-walled
epithelium
base.
cavernous
der of cow, from the state
epithelium
X 100.
in urinary
calf which was inoculated
present
fibromatous
cells.
areas
of
non
material
vascular
81
channels
was in junction
with
the
X 110.
hemanginoma
of Washington,
in urinary
with enzootic
blad
hematuria.
The thin-walled vascular channels are similar to those in the ex
perimentally induced lesion illustrated in Fig. 10. X 300.
FIG. 12.—Papillary
Japan
with
enzootic
adenoma
hematuria.
in urinary
bladder
of cow from
The
cyst-like
spaces
around
periphery of the lesion were lined with columnar
Fibrous connective tissue was present in submucosa.
sue section
through
courtesy
of 1)r. Yutaka
epithelium.
X 6. (Tis
Fujimoto,
Hokaido
Univ., Japan.)
FIG.
13.—Transitional
cell
adenocarcinoma
in urinary
bladder
of COWfrom Turkey with enzootic hematuria.
Benign glandular
metaplasia
in the epithelium.
Dense
(Tissue
and malignant
changes
were present
fibromatous
tissue
was present
in the submucosa.
section
through
courtesy
of Dr. M. Pamukcu,
X 50.
Univ.
of
Ankara, Turkey.)
16
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
@
-
-
:
:@c.
9
@
.@%.
.•@
,.
. ..
-..@.
. .
-
-.
._‘\@‘
@.. -.
.1
•
-a..'.
•@•@
. -
4'
‘1
P 7 -,
p.
-‘
17
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
BROBST
of the bovine bladder (2). The mucosal projections
produced by the papilloma agent were, however, more
massive and the reaction could appropriately be considered
polypoid
hyperplasia.
Papillary
hyperplasia
is believed
to be a response to an inflammatory reaction or chronic
irritation
which
produces
edema
in the
bladder
sub
mucosa and causes a proliferation of the bladder mucosa
(6).
In the
experimentally
induced
tumors
the
fibroma
tous mass in the bladder wall may have supplied the
stimulus
necessary
for this process to occur.
The number and length of the mucosal projections were
related directly
to the size of the fibromatous
mass in the
tumor base and its depth of penetration into the bladder
wall.
This observation
was most evident
in tumors
of
vaccinated calves. The mucosal projections of these
tumors were few and small; similarly, the fibromatous
reaction in the wall of the bladder was smaller than in
nonvaccinated calves. Immunization would seem to limit
the extent of the fibrobla.stic reaction and thereby might
limit accompanying inflammatory response. Age of the
tumor was also a factor determining the size of the mu
cosal polyps. The longest polyps were in tumors older
than 120 days. However, in those tumors in which fibrosis
of the polyps was extensive, cystoscopic examinations
prior to necropsy indicated that the polyps had ceased
growing in length and in number.
Although
the primary lesion in the experimentally
induced tumor was due to a proliferation of fibroblasts in
the submucosa of the bladder, the transitional epitheium
displayed
alterations
marked changes.
The sequence of epithelial
in areas of polypoid hyperplasia was a thicken
ing followed by glandular metaplasia, and often the de
velopment of Brunn's nests. Glandular metaplasia was
not observed in tumors less than 60 days of age but ap
peared to increase progressively in older tumors. Glandu
lar metaplasia in tumors more than 180 days old was simi
lar to naturally occurring glandular metaplasia of the
bovine bladder, although growths resembling mucinous
adenomas of the bladder were not present (2). Squamous
metaplasia
was observed in tumors of various ages.
Simi
lar epithelial alterations have been observed in the urinary
bladder of man (3, 6, 10). Cattle with enzootic hematuria
have glandular and squamous metaplasia of the bladder
epithelium
19
AND OLSON—Bladder Tumors and a Papilloma Agent
as well as hemangiomatous
areas and fibroma
tous changes in the submucosa (Figs. 11—13)similar to
the bladder lesions produced experimentally with cutane
ous papilloma material. Progressive neoplasia of epi
thelial or mesenchymal elements as seen in enzootic hema
turin (4, 9) was not observed in our experimentally induced
lesions
bladder
change
others
chronic
in the
which did regress.
Glandular metaplasia in the
of man is regarded by some as a precancerous
which can progress to adenocarcinoma,
although
consider it simply cellular metapla.sia secondary to
irritation
(3, 10). The similar lesion observed
present experiments may be different or it may
require some additional stimulus for malignancy.
Mar
tincic (4) observed in Yugoslavia that half the cattle
which had to be slaughtered because of hematuria and 80%
of those that died from the disease had pronounced malig
nant alterations
strongly
of the urinary
suggests
an additional
bladder
epithelium.
This
factor which requires
time
for its action.
REFERENCES
1. BAGDONAS,
V., ANDOLSON,C., JR. Observations on Immunity
in
Cutaneous
Bovine
Papillomatosis.
Am.
J.
Vet.
Res.,
15:
240—45,1954.
2. BROBST,D. F., ANDOLSON,C. Neoplastic and Proliferative
Lesions
of the
Bovine
Urinary
Bladder.
Am.
J. Vet.
Res.,
24:
105—11,1963.
3. EMMETT, J. L., AND MCDONALD, J. R. Proliferation
of Glands
of the Urinary
Bladder
Simulating
Malignant
Neoplasm.
J.
Urol., 48: 257-65, 1942.
4. [email protected],M. Krvaco mokrenje goveda. Haematuria vesi
calis cancerogenes bovis. Veterinaria, 4: 525—620,1955.
5. MCENTEE, K. Fibropapillomas
of the External
Genitalia
Cattle. Cornell Vet., 40: 304—12,1950.
6. MOsT0FI, F. K. Potentialities of Bladder Epithelium.
of
J. Urol.,
71: 705—14,
1954.
7. OLSON, C., LUEDKE, A. J., AND BROBST, D. F. Induced
In
munity of Skin, Vagina and Urinary Bladder to Bovine Papil
lomatosis. Cancer Res., 22: 463—68,1962.
8. OLSON, C., PAMUKCU,A. M., BROBST, D. F., KOWALCZYK,T.,
SATTER,
E.
J.,
AND PRICE,
J. M.
A Urinary
Bladder
Tumor
Induced by a Bovine Cutaneous Papilloma Agent. Cancer
Res., 19: 779—82,1959.
9. PAMUKCU,A. M. Tumors of the Urinary Bladder in Cattle
and Water Buffalo Affected with Enzootic Bovine Hematuria.
Zentr. Veterinaermed.,
4: 185—97,1957.
10. PATCH, F. S., ANDRHEA, L. J. The Genesis and Development
of Brunn's Nests and Their Relation to Cystitis Cystica,
Cystitis Glandularis and Primary Adeno-carcinoma
of the
Bladder. Can. Med. Assoc. J., 33: 597—606,1935.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.
Histopathology of Urinary Bladder Tumors Induced by Bovine
Cutaneous Papilloma Agent
D. F. Brobst and C. Olson
Cancer Res 1965;25:12-19.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/25/1_Part_1/12
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1965 American Association for Cancer Research.