0300985811419534

Prévia do material em texto

Loss of Retinoblastoma Protein, But Not
p53, Is Associated With the Presence of
Papillomaviral DNA in Feline Viral Plaques,
Bowenoid In Situ Carcinomas, and
Squamous Cell Carcinomas
J. S. Munday1 and D. Aberdein1
Abstract
Although papillomaviral (PV) DNA is frequently present in feline cutaneous squamous cell carcinomas (SCCs), a causative
association cannot be proven. Oncogenic human PVs cause neoplastic transformation by inhibiting retinoblastoma (pRb) and p53
activity. Therefore, absence of pRb and p53 immunostaining, along with increased p16 immunostaining, indicates a PV cause in
some human SCCs. If PVs cause cutaneous feline SCCs, it was hypothesized that a similar immunohistochemistry profile, along
with PV DNA, would be detectable. This was investigated using 5 feline viral plaques, 10 Bowenoid in situ carcinomas, 19 SCCs
from ultraviolet-exposed (UV-exposed) skin, and 11 SCCs from UV-protected skin. Papillomaviral DNA was amplified by poly-
merase chain reaction from 30 of 45 lesions. Reduced pRb immunostaining was present in 26 of 45; increased p16 immunostaining
was in 30; and p53 immunostaining was in 19. Both reduced pRb immunostaining and increased p16 immunostaining were more
frequent in lesions containing PV DNA. In contrast, no association was observed between p53 immunostaining and the presence
of PV DNA. SCCs from UV-protected skin more frequently contained PV DNA, reduced pRb, and increased p16 than
UV-exposed SCCs. UV exposure was not associated with p53 immunostaining within the SCCs. These results suggest that feline
PVs alter cell regulation by degrading pRb. Unlike oncogenic human PVs, there was no evidence that feline PVs degrade p53. These
results provide further evidence that PVs may cause feline cutaneous SCCs, especially those in UV-protected skin, and they
suggest a possible mechanism of this oncogenic action.
Keywords
squamous cell carcinoma, Bowenoid in situ carcinoma, cat, papillomavirus, retinoblastoma, p16, p53, ultraviolet light, viral
oncogenesis, skin
Squamous cell carcinomas (SCCs) are the most common
malignant skin neoplasm of cats.16 While ultraviolet (UV)
exposure is considered to cause most feline cutaneous
SCCs,16 papillomaviral (PV) DNA can be detected within a
proportion of neoplasms, most frequently those from UV-
protected areas of the body.23 Although this detection of
PV DNA suggests a possible causal association between PVs
and feline cutaneous SCCs, PV DNA can be detected within
non-SCC feline skin samples, making it difficult to determine
whether the PVs cause SCC development or are present as
‘‘innocent bystanders.’’23
In humans, PVs are an established cause of genital and oral
SCCs.33 Oncogenic human papillomaviruses (HPVs) cause
neoplastic transformation by inactivating and degrading both
the retinoblastoma protein (pRb), which subsequently increases
p16CDKN2A protein, and the transformation-related protein 53
(p53).5,11,34,35 This action of HPVs is so consistent that immu-
nohistochemistry can be used to determine if a oral SCC is
caused by HPV infection.6,36 A HPV-induced SCC demon-
strates an absence of pRb and p53 immunostaining with an
increase in p16.10,20 In contrast, a SCC that develops due to
chronic UV exposure typically demonstrates p53 and pRb
immunostaining without an increase in p16.8,32
The purpose of the present experiment was to determine
whether the presence of PV DNA within a feline cutaneous
SCC is associated with a immunohistochemical profile similar
to that seen in PV-induced human cancers. Therefore, it was
hypothesized that feline cutaneous SCCs could be divided into
1Institute of Veterinary, Animal and Biomedical Sciences, Massey University,
Palmerston North, New Zealand
Corresponding Author:
John S. Munday, Institute of Veterinary, Animal and Biomedical Sciences,
Massey University, Palmerston North 4442 New Zealand
Email: j.munday@massey.ac.nz
Veterinary Pathology
49(3) 538-545
ª The Author(s) 2012
Reprints and permission:
sagepub.com/journalsPermissions.nav
DOI: 10.1177/0300985811419534
http://vet.sagepub.com
2 groups. In the first group, neither pRb nor p53 immunos-
taining would be visible, but increased p16 and PV DNA
would be present. This immunohistochemical prolife would
suggest a PV cause, and these SCCs would be mostly from
UV-protected areas of the body. The second group would
contain pRb and p53 immunostaining without any increased
p16 immunoreactivity, more consistent with the SCCs being
UV induced. SCCs with this immunohistochemical profile
would occur more commonly in UV-exposed skin and would
less frequently contain PV DNA. As they are believed to be
caused by PV infection,24,26,29,38,41 feline viral plaques and
Bowenoid in situ carcinomas (BISCs) were included with
SCCs from UV-exposed and UV-protected areas of skin.
Polymerase chain reaction was used to detect PV DNA
within all lesions. To our knowledge, this is the first time that
antibodies against human pRb have been validated and used
in a nonlaboratory animal species.
Material and Methods
Feline viral plaques, BISCs, and invasive SCCs were identified
by retrospective surveys of databases at Massey University,
New Zealand Veterinary Pathology Ltd, and Gribbles Veterin-
ary Pathology Ltd. The diagnosis was confirmed with histolo-
gic examination of an hematoxylin and eosin–stained section
based on previously described criteria.16 For the purpose of the
study, locations on the body were subdivided into UV exposed,
if the area contained little protective hair, whereas densely
haired areas were considered UV protected. Therefore, SCCs
on the pinna, nasal planum, eyelid, or third eyelid were consid-
ered UV exposed while UV-protected SCCs were from the
face, digit, thigh, or neck.
DNA was extracted from formalin-fixed, paraffin-
embedded samples as previously described.25 Four primers sets
were used to amplify PV DNA from the DNA extracted from
the samples. These included the MY09/11 consensus primers
that have been shown to amplify PV DNA from multiple spe-
cies9 and the JMPF/R primers that amplify Felis domesticus
papillomavirus 2 (FdPV-2) DNA.24 The JMY2F/R and
JMY3F/R primers were used to amplify 2 PV DNA sequences
that have been detected in feline cutaneous SCCs.22 All poly-
merase chain reactions were carried out in duplicate. DNA
extracted from a feline viral plaque was used as a positive con-
trol for the JMPF/R primers, while DNA extracted from a
bovine fibropapilloma was used for the MY09/11 primers.
DNA extracted from a bovine fibropapilloma was used as a
negative control for the JMPF/R, JMY2F/R, and JMY3F/R pri-
mers, while the negative control for the MY09/11 primers did
not contain template DNA. DNA amplified by the MY09/11
primers was purified and sequenced as previously described.25
Results were compared with known sequences in GenBank
(http://www.ncbi.nlm.nih.gov/genbank) using the basic local
alignment search tool (http://www.ncbi.nlm.nih.gov/blast). Due
to the previously demonstrated specificity of the JMPF/R,
JMY2F/R, and JMY3F/R primers, the DNA amplified by these
primers was not sequenced.22,24
As antibodies against pRb have not been used in cats,
Western blotting was used to confirm the specificity of the anti-
bodies used to feline pRb. Briefly, tissue lysates of feline liver
were prepared and diluted in Laemmli buffer to a final protein
concentration of 2.5 mg/mL and heated at 100�C for 5 minutes.
A Jurkat cell lysate (BD Biosciences, San Jose, CA) was used
as a positive control. Samples were loaded onto a SDS-PAGE
7.5% gel (Criterion Precast Tris-HCl Gel, BioRad, Hercules,
CA) and separated by electrophoresis at 100 V for 60 minutes
in 1� Tris-glycine-SDS buffer. Proteins were transferred
ontonitrocellulose membrane, and nonspecific binding was
blocked by incubation in 5% skimmed milk in PBS/Tween
20 for 1 hour at room temperature. Membranes were incubated
overnight at 4�C with mouse anti-human pRb (BD Bios-
ciences) at a dilution of 1:100. Membranes were incubated with
horseradish peroxidase–conjugated secondary antibodies (BD
Biosciences) for 1 hour at room temperature, and immune com-
plexes were visualized using a chemiluminescent detection
system (Immun-Star HRP Chemiluminescent Kit, BioRad).
Sections for immunohistochemistry were cut at a thickness
of 5 mm, deparaffinized in xylene, rehydrated in graded etha-
nol, and rinsed in distilled water. To perform immunohisto-
chemistry to detect pRb, sections were heated in a pressure
cooker for 10 minutes in 0.01M citrate buffer, pH 6.0, with a
10-minute cooldown. Endogenous peroxidases were blocked
by incubating slides in 0.3% hydrogen peroxide in 0.05M
Tris-buffered saline, pH 7.6. Nonspecific staining was blocked
by incubating slides with equine serum (Vector Laboratories,
Burlingame, CA) for 20 minutes. Slides were incubated for
60 minutes with anti-human pRb antibodies (BD Biosciences)
at a dilution of 1:40. Slides were incubated for 30 minutes
with biotinylated horse anti-mouse/rabbit secondary antibody
(Vector Laboratories). Slides were then incubated for 30 min-
utes with a biotin–avidin complex (Vector Laboratories), fol-
lowed by 3,3-diaminobenzidine substrate (Liquid DAB
Substrate Chromagen System, Dako, Carpinteria, CA) and
counterstained with Gill’s hematoxylin. Cells within the adja-
cent nonneoplastic epidermis were used as internal positive
controls, while the primary antibody was omitted in negative
controls.
Immunohistochemistry to detect p16 was based on a mouse
anti-human p16 monoclonal antibody (BD Biosciences) at a
dilution of 1:200 as previously reported.21 Epithelial cells
within the dermal papilla exhibited consistent low-intensity
cytoplasmic positivity and were used as an internal positive
control, while the epidermis surrounding the lesion was used
as a negative control.
To detect p53, a mouse anti-human p53 clone pAb 240 anti-
body (BD Biosciences) was used as previously reported1
except that the antibody was diluted to 1:100. A human neo-
plasm known to contain p53 mutations was used as a positive
control, and the epidermis surrounding the lesion was used as
a negative control. The pAb 240 p53 clone was used because
it has also been reported to produce less background staining
than the CM-1 p53 clone, which has been reported to react with
feline p53.1
Munday and Aberdein 539
For all examined antibodies, the percentage of cells with
nuclear immunoreactivity was estimated by examining 5 dif-
ferent 400� fields within each lesion. Lesions were also
assessed to determine if immunoreactivity was distributed
evenly within the neoplasm. Lesions were considered to con-
tain increased p16 when greater than 75% of the cells contained
intense immunostaining. When pRb was assessed, lesions were
divided into immunostaining visible in less than 25% of the
cells, in 25% to 50% of the cells, and in over 50% of the cells;
pRb was considered to be reduced within a lesion if less than
half the cells contained immunostaining. Lesions were classi-
fied as having immunostaining against p53 if intense immunos-
taining was visible within over 20% of the cells. Differences
between groups were investigated by analysis of variance using
SPSS 16 (SPSS Inc, Chicago, IL).
Results
Forty-five samples were included in this study, including 5
feline viral plaques, 10 BISCs, and 30 invasive SCCs. Of the
invasive SCCs, 19 were from UV-exposed skin, including 8
from the pinnae, 6 from the nasal planum, 4 from the eyelids,
and 1 from the third eyelid. Eleven SCCs were from UV-
protected skin, including 5 from the face, 2 from the lips, 2
from the digits, and 1 each from the ventrum and neck.
The 4 sets of primers used in the experiment amplified PV
DNA from 30 samples, including all viral plaques and BISCs
(Table 1). Of 30 SCCs, 15 contained PV DNA. PV DNA was
detected more frequently in SCCs from UV-protected areas
(9 of 11) than in UV-exposed SCCs (6 of 19) (P ¼ .01). Of the
30 samples that contained PV DNA, 29 contained DNA that
was amplified by the primers specific for FdPV-2. One SCC
from a UV-protected area contained only the previously
described FdPV-MY2 DNA sequence.26 Both the FdPV-MY2
sequence and FdPV-2 were detected in 2 SCCs and a BISC.
One viral plaque contained FdPV-2 and the previously
described FdPV-MY1 DNA sequence,27 while one SCC con-
tained FdPV-2 and the previously described FdPV-MY3 DNA
sequence.22
Western blotting using anti-pRb antibodies demonstrated a
110-kDA fragment from the positive control and feline tissues,
confirming the specificity of this antibody for feline pRb.
Immunoreactivity using anti-pRb antibodies was confined to
the nucleus within all samples. Within the 45 lesions, pRb
immunoreactivity was visible within less than 25% of the cells
in 18 lesions: 3 viral plaques, 5 BISCs, and 10 SCCs (Figs. 1–
3). Immunoreactivity was present in 25% to 50% of the cells
within 2 viral plaques, 3 BISCS, and 3 SCCs. Therefore,
reduced pRb immunoreactivity was observed in 26 lesions,
including all 5 viral plaques, 8 BISCs, and 13 SCCs. Reduced
pRb immunoreactivity was more frequent in lesions that con-
tained PV DNA (24 of 30) than in lesions without detectable
PV DNA (2 of 15) (P < .001) (Table 2). Within the 30 SCCs,
p53 immunostaining was not visible more frequently in SCCs
from UV-exposed skin (10 of 19) than in SCCs from UV-pro-
tected skin (4 of 11) (p ¼ 0.32).
Immunoreactivity against p16 was present within both
the nuclei and the cytoplasm. All lesions contained either
marked immunoreactivity within most cells or minimal
immunoreactivity within less than 25% of the cells. Intense
immunoreactivity was present within all viral plaques and
BISCs and within 15 SCCs. Increased p16 was present more
frequently in lesions that contained PV DNA (28 of 30)
than in lesions without detectable PV DNA (2 of 15) (P <
.001). Within the SCCs, increased p16 was more frequent
in SCCs from UV-protected sites (10 of 11) than from
UV-exposed sites (5 of 19) (P ¼ .001). Of the 30 lesions
that contained increased p16, loss of pRb immunoreactivity
was present within 26 (Fig. 4). No lesions contained
reduced pRb immunostaining without a concurrent increase
in p16 immunostaining.
Immunostaining using anti-p53 antibodies was confined to
the nuclei. Immunoreactivity was visible within 19 of 45
lesions—5 BISCs and 14 SCCs (Figs. 5, 6). There was no sig-
nificant difference in the frequency of p53 immunostaining
between lesions that contained PV DNA (12 of 30) and those
that did not (7 of 15) (P ¼ .45). There were no significant dif-
ferences in p53 immunostaining between lesions with reduced
pRb (P ¼ .183) or those with increased p16 (P ¼ .227). Within
the 30 SCCs, more SCCs from UV-exposed skin (10 of 19) did
not contain p53 immunostaining than SCCs from UV-protected
skin (4 of 11) (P ¼ .32).
Table 1. Polymerase Chain Reaction and Immunohistochemistry Results, No. (%)a
Lesion Total PV DNA Detected Loss of pRbb Increased p16c Presence of p53d
Feline viral plaque 5 5 (100) 5 (100) 5 (100) 0 (0)
Bowenoid in situ carcinoma 10 10 (100) 8 (80) 10 (100) 5 (50)
SCCs 30 15 (50) 13 (43) 15 (50) 14 (47)
UV-exposed SCCse 19 6* (32) 4* (21) 5* (26) 10 (53)
UV-protected SCCsf 11 9* (82) 9* (82) 10* (91) 4 (36)
a PV, papillomaviral; SCC, squamous cell carcinoma; UV, ultraviolet.
b Indicates that nuclear immunostaining using anti-retinoblastoma protein antibodies was present in less than 50% of theneoplastic cells.
c Indicates that greater than 75% of the neoplastic cells demonstrated immunoreactivity against anti-p16CDKN2A protein antibodies.
d Indicates that greater than 20% of the neoplastic cells demonstrated immunoreactivity against anti-transformation-related protein 53 antibodies.
e From the pinna, nasal planum, eyelid, or third eyelid.
f From the face, digit, thigh, or neck.
* P < .01, between UV-protected and UV-exposed SCCs.
540 Veterinary Pathology 49(3)
Figure 1. Feline viral plaque. Cells within the epidermis show cytopathologic changes consistent with papillomavirus infection, including enlar-
gement and cytoplasmic vacuolation. Cells showing cytopathologic changes contain loss of nuclear immunoreactivity against anti-retinoblastoma
protein antibodies. Keratinocytes within the surrounding normal epidermis have retained nuclear immunostaining (arrows). Avidin–biotin–per-
oxidase with hematoxylin counterstain. Figure 2. Feline Bowenoid in situ carcinoma. Keratinocytes within the proliferating epidermis do not
contain nuclear immunoreactivity against anti-retinoblastoma antibodies. In contrast, surrounding nonneoplastic keratinocytes retain nuclear
immunostaining using this antibody (arrows). Avidin–biotin–peroxidase with hematoxylin counterstain. Figure 3. Feline invasive squamous cell
carcinoma from the face. Nuclear immunoreactivity against anti-retinoblastoma antibodies is lost throughout the majority of the neoplastic cells.
Munday and Aberdein 541
Discussion
In cats, there is strong evidence that PVs cause viral plaques
and BISCs.24,26,29,41 Viral plaques can progress to BISCs,41
and BISCs have been reported to progress to SCCs,16 but the
proportion of feline SCCs that develop from BISCs and the role
that PVs play in this progression are unresolved. PV DNA was
amplified from 50% of the SCCs in the present study. However,
as PVs can asymptomatically infect feline skin,28 the detection
of PV DNA within a cutaneous SCC does not prove that the
virus influenced neoplasm development. As has been
reported,22 PV DNA was detected in the present study more
frequently in SCCs from UV-protected areas than in SCCs
from UV-exposed skin. In people, protection from UV light
decreases asymptomatic PV skin infection.14 This suggests that
the increased detection of PV DNA within feline cutaneous
SCCs that were protected from UV light was not due to an
increased frequency of asymptomatic infection.
As with cats, many human PV infections are asymptomatic,3
making it difficult to determine the significance of HPV DNA
within a neoplasm. To help differentiate between an HPV cause
and an asymptomatic infection, immunohistochemistry can be
used to detect changes to cell regulatory proteins that are
caused by HPVs. One of these is the HPV-induced inactivation
and degradation of pRb.5,11 The pRb inhibits progression from
the G1 phase to the S phase of the cell cycle, and loss of pRb
removes an important checkpoint preventing cell division.37
This mechanism of neoplastic transformation is so consistent
that demonstration of reduced pRb within an oral or anogenital
SCC is considered indicative of a HPV cause.2,20,40 In the pres-
ent study, reduced pRb immunostaining was present in 26 of
the 45 feline preneoplastic and neoplastic lesions. Of the 26
lesions that demonstrated reduced pRb, PV DNA was ampli-
fied from 24. As in previous studies,24,30 FdPV-2 was the PV
most frequently amplified from the feline lesions. The ability
of HPVs to interact with pRb is dependent on a LXCXE
motif within the E7 gene.11 This motif has been identified in
FdPV-2.18 Therefore, the significant association between the
presence of PV DNA and reduced pRb suggests that FdPV-2
can bind to and degrade pRb. Within the PVs that infect nonhu-
man species, only the cottontail rabbit papillomavirus has been
shown to degrade pRb.15,17 However, this experiment was
performed in cell cultures, and to our knowledge, an associa-
tion between PV infection and loss of pRb has not been demon-
strated in samples of naturally developing lesions from a
nonhuman species.
PV DNA was not amplified from 2 of the 26 lesions that
contained reduced pRb immunostaining. This could suggest
that loss of pRb is not specific for a PV cause in feline cuta-
neous SCCs. Alternatively, it is possible that infection by a
PV had reduced pRb, but the PV was not detected within the
SCC. Multiple PV types, including HPVs, have been detected
within feline cutaneous SCCs.25,29,30 Therefore, while consen-
sus primers were used in this study, it is possible that other PV
types were present that were not amplified by these primers.
Additionally, the tissues used in the study had been formalin
fixed. Such fixation can fracture DNA, making amplification
difficult, especially if only small quantities of PV DNA are
present.19
In humans, the presence of increased p16 immunostaining is
also used to indicate a PV causewithin SCCs from some anatomi-
cal locations.10,20 As p16 regulates cell division by a mechanism
that is dependent on the presence of pRb,37 loss of pRb increases
p16 within the cell.34 In the present study, all feline lesions with
reduced pRb immunostaining also contained increased p16
immunostaining, suggesting that inactivation of pRb consistently
increased p16 within the feline lesions. However, 2 SCCs and 2
BISCs contained increased p16 without reduced pRb immunos-
taining. While this could be evidence that p16 can increase inde-
pendently of pRb, studies ofHPVs suggest that the primary action
of the PV is to inhibit pRb function, with accelerated protein
degradation as a secondary function.5,11 Therefore, pRb can
remain immunohistochemically detectable within human PV-
induced SCCs despite being functionally inactive.20Associations
between increased p16 and the presence of PVDNAwithin feline
cutaneous SCCs have been reported.21,22However, this is the first
evidence to suggest that feline PV infection could increase p16 by
degrading pRb. While these results suggest that feline PV infec-
tion may alter cellular regulation by disrupting the pRb/p16 path-
way, it cannot be confirmed that this action influences neoplastic
transformation of the cells.
In humans, a PV-induced SCC is expected to contain PV
DNA with reduced pRb, and increased p16, immunostaining.
Table 2. Presence of Papillomaviral DNA Within a Lesion, No. (%)a
Lesions Total
Loss of
pRbb
Increased
p16c
Presence of
p53d
With PV DNA 30 24* (80) 28* (93) 12 (40)
Without PV
DNA
15 2* (13) 2* (13) 7 (47)
a PV, papillomaviral.
b Indicates that nuclear immunostaining using anti-retinoblastoma protein
antibodies was present in less than 50% of the neoplastic cells.
c Indicates that greater than 75% of the neoplastic cells demonstrated
immunoreactivity against anti-p16CDKN2A protein antibodies.
d Indicates that greater than 20% of the neoplastic cells demonstrated
immunoreactivity against anti-transformation-related protein 53 antibodies.
* P < .01, between lesions that contained PV DNA and those that did not.
Figure 3 (continued) Avidin–biotin–peroxidase with hematoxylin counterstain. Figure 4. The same squamous cell carcinoma as Figure 3.
Prominent nuclear and cytoplasmic immunoreactivity against the p16CDKN2A protein is visible within neoplastic cells. Bond Refine Detection
staining kit. Figure 5. Feline Bowenoid in situ carcinoma. Prominent nuclear immunoreactivity against anti-p53 antibodies is visible within the
lesion. Avidin–biotin–peroxidase with hematoxylin counterstain. Figure 6. Feline invasive squamous cell carcinoma from the pinna. Nuclear
immunoreactivity using anti-p53 antibodies is prominent within the neoplastic cell population. Avidin–biotin–peroxidase with hematoxylin
counterstain.542 Veterinary Pathology 49(3)
In the present study, all PV-induced plaques and BISCs con-
tained PV DNA and increased p16 with reduced pRb visible
in 13. PV DNA with reduced pRb and increased p16 immunos-
taining was also observed in 11 SCCs. This suggests that 11 of
the 30 SCCs in the present study were caused by PV infection.
Nine SCCs with this profile developed in UV-protected skin,
suggesting that they may have developed as a progression of
a PV-induced BISC.
The strong correlation between pRb loss and increased p16
observed in the present study suggests that it is unnecessary to
evaluate both proteins to detect changes in the pRb/p16 path-
way. Immunoreactivity against p16 was biphasic within the
feline lesions, making determination of p16 status quick and
easy. In contrast, detection of reduced pRb by immunohisto-
chemistry required estimating the proportion of cells that
retained pRb immunostaining and so was more time-
consuming. Therefore, immunohistochemistry to detect p16
appears to be a better method to detect changes in the pRb/
p16 pathway within feline samples. In human oral SCCs,
immunohistochemistry to detect p16, rather than pRb, is cur-
rently used (along with molecular techniques) to investigate a
PV cause.6,36
The p53 protein functions as a tumor suppressor, maintain-
ing genetic integrity and promoting apoptosis in response to
DNA damage.37 Mutations within the p53 gene prevent normal
p53 function and result in a stable p53 protein that is detectable
immunohistochemically.13,42 Oncogenic HPVs degrade
p537,31,35 so that p53 immunostaining is not present in human
PV-induced SCCs.20 In contrast, in the present study, there was
no significant association between p53 immunostaining and the
presence of PV DNA within the lesions, which suggests that
feline PVs do not degrade p53. This is consistent with bovine
PV-induced equine sarcoids that also demonstrate p53 immu-
nostaining in a proportion of tumors.4 Likewise, no interaction
between cottontail rabbit papillomavirus proteins and p53
could be detected.15 In the present study, immunostaining
against p53 was visible in 5 of 10 BISCs. A previous study
of 22 BISCs detected p53 immunostaining in just 4.12 The rea-
sons for the different rates of p53 immunoreactivity are uncer-
tain. However, it is possible that p53 mutations develop
spontaneously as dysplasia within the BISC increases. This is
supported by the absence of p53 immunostaining and dysplasia
within the 5 viral plaques. If p53 mutations develop as a pre-
neoplastic lesion progresses to neoplasia, it is possible that the
BISC lesions included in the present study were more advanced
than the BISC lesions included in the previous study.12
In the present study, p53 immunostaining was present
within 14 of 30 cutaneous SCCs. Previous studies of feline
SCCs have detected p53 in 13 of 20 cutaneous feline SCCs39
and 7 of 9 feline cutaneous and oral SCCs.1 In people, UV
exposure causes p53 gene mutations, and p53 immunostaining
is frequently present in human UV-induced skin cancers.32
In cats, p53 immunostaining has been associated with UV
exposure in preneoplastic cutaneous lesions.12 In contrast, no
significant association between UV exposure and p53 immu-
nostaining was observed in the present series of feline SCCs.
This could be due to the development of additional mutations
within the p53 gene that result in the loss of immunostaining
within some SCCs and the development of immunostaining
in others.
In conclusion, the presence of PV DNA within the feline
preneoplastic and neoplastic cutaneous lesions was associated
with reduced pRb immunostaining. This suggests that, similar
to oncogenic HPVs, feline PVs may influence cell regulation
by inactivating and degrading the pRb protein. Reduced pRb
was associated with increased p16 within the neoplastic cells,
and p16 immunostaining appears to be a reliable indicator of
reduced pRb function within a cell. Loss of pRb and, subse-
quently, increased p16 is considered a specific indicator of a
PV cause in human oral and anorectal SCCs.10,20 If loss of pRb
within feline lesions similarly indicates a PV cause, the results
of the present study suggest that PV infection could cause most
feline SCCs from UV-protected skin and a significant propor-
tion of UV-exposed SCCs. The results of this study provide
additional evidence that the pathogenesis of feline cutaneous
SCCs shows some similarities to the pathogenesis of human
cervical and oral SCCs. However, in contrast to oncogenic
HPVs, feline PVs do not appear to degrade p53.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with
respect to the research, authorship, and/or publication of this
article.
Funding
The research was supported by a grant from the Massey
University Research Fund.
References
1. Albaric O, Bret L, Amardeihl M, et al. Immunohistochemical
expression of p53 in animal tumors: a methodological study using
four anti-human p53 antibodies. Histol Histopathol. 2001;16:
113–121.
2. Andl T, Kahn T, Pfuhl A, et al. Etiological involvement of onco-
genic human papillomavirus in tonsillar squamous cell carcino-
mas lacking retinoblastoma cell cycle control. Cancer Res.
1998;58:5–13.
3. Antonsson A, Forslund O, Ekberg H, et al. The ubiquity and
impressive genomic diversity of human skin papillomaviruses
suggest a commensalic nature of these viruses. J Virol. 2000;
74:11636–11641.
4. Bogaert L, Van Poucke M, De Baere C, et al. Bovine papilloma-
virus load and mRNA expression, cell proliferation and p53
expression in four clinical types of equine sarcoid. J Gen Virol.
2007;88:2155–2161.
5. Boyer SN, Wazer DE, Band V. E7 protein of human papilloma
virus-16 induces degradation of retinoblastoma protein
through the ubiquitin-proteasome pathway. Cancer Res. 1996;
56:4620–4624.
6. Braakhuis BJ, Brakenhoff RH, Meijer CJ, et al. Human papilloma
virus in head and neck cancer: the need for a standardised assay to
Munday and Aberdein 543
assess the full clinical importance. Eur J Cancer. 2009;45:
2935–2939.
7. Braakhuis BJ, Snijders PJ, Keune WJ, et al. Genetic patterns in
head and neck cancers that contain or lack transcriptionally active
human papillomavirus. J Natl Cancer Inst. 2004;96:998–1006.
8. Brash DE, Ziegler A, Jonason AS, et al. Sunlight and sunburn in
human skin cancer: p53, apoptosis, and tumor promotion. J Inves-
tig Dermatol Symp Proc. 1996;1:136–142.
9. Chan SY, Delius H, Halpern AL, et al. Analysis of genomic
sequences of 95 papillomavirus types: uniting typing, phylogeny,
and taxonomy. J Virol. 1995;69:3074–3083.
10. Cunningham LL, Jr, Pagano GM, Li M, et al. Overexpression of
p16INK4 is a reliable marker of human papillomavirus-induced
oral high-grade squamous dysplasia. Oral Surg Oral Med Oral
Pathol Oral Radiol Endod. 2006;102:77–81.
11. Dyson N, Howley PM, Munger K, et al. The human papilloma
virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene
product. Science. 1989;243:934–937.
12. Favrot C, Welle M, Heimann M, et al. Clinical, histologic, and
immunohistochemical analyses of feline squamous cell carci-
noma in situ. Vet Pathol. 2009;46:25–33.
13. Finlay CA, Hinds PW, Tan TH, et al. Activating mutations for
transformation by p53 produce a gene product that forms an
hsc70–p53 complex with an altered half-life. Mol Cell Biol.
1988;8:531–539.
14. Forslund O, Iftner T, Andersson K, et al. Cutaneous human papil-
lomaviruses found in sun-exposed skin: Beta-papillomavirus spe-
cies 2 predominates in squamous cell carcinoma. J Infect Dis.
2007;196:876–883.
15. Ganzenmueller T, Matthaei M, Muench P, et al.The E7 protein of
the cottontail rabbit papillomavirus immortalizes normal rabbit
keratinocytes and reduces pRb levels, while E6 cooperates in
immortalization but neither degrades p53 nor binds E6AP. Virol-
ogy. 2008;372:313–324.
16. Gross TL, Ihrke PJ, Walder EJ, et al. Skin Diseases of the Dog and
Cat: Clinical and Histopathologic Diagnosis. 2nd ed. Oxford,
UK: Blackwell Science; 2005:571–574.
17. Haskell KM, Vuocolo GA, Defeo-Jones D, et al. Comparison of
the binding of the human papillomavirus type 16 and cottontail
rabbit papillomavirus E7 proteins to the retinoblastoma gene
product. J Gen Virol. 1993;74:115–119.
18. Lange CE, Tobler K, Markau T, et al. Sequence and classification
of FdPV2, a papillomavirus isolated from feline bowenoid in situ
carcinomas. Vet Microbiol. 2009;137:60–65.
19. Little SE, Vuononvirta R, Reis-Filho JS, et al. Array CGH
using whole genome amplification of fresh-frozen and formalin-
fixed, paraffin-embedded tumor DNA. Genomics. 2006;87:
298–306.
20. Lu DW, El-Mofty SK, Wang HL. Expression of p16, Rb, and p53
proteins in squamous cell carcinomas of the anorectal region har-
boring human papillomavirus DNA. Mod Pathol. 2003;16:
692–699.
21. Munday JS, French AF, Peters-Kennedy J, et al. Increased
p16CDKN2A protein within feline cutaneous viral plaques,
bowenoid in situ carcinomas, and a subset of invasive squamous
cell carcinomas. Vet Pathol. 2011;48:460–465.
22. Munday JS, Gibson I, French AF. Papillomaviral DNA and
increased p16(CDKN2A) protein are frequently present within
feline cutaneous squamous cell carcinomas in ultraviolet-
protected skin. Vet Dermatol. 2011;22:360–366.
23. Munday JS, Kiupel M. Papillomavirus-associated cutaneous neo-
plasia in mammals. Vet Pathol. 2010;47:254–264.
24. Munday JS, Kiupel M, French AF, et al. Amplification of papil-
lomaviral DNA sequences from a high proportion of feline cuta-
neous in situ and invasive squamous cell carcinomas using a
nested polymerase chain reaction. Vet Dermatol. 2008;19:
259–263.
25. Munday JS, Kiupel M, French AF, et al. Detection of papilloma-
viral sequences in feline Bowenoid in situ carcinoma using con-
sensus primers. Vet Dermatol. 2007;18:241–245.
26. Munday JS, Peters-Kennedy J. Consistent detection of Felis
domesticus papillomavirus-2 DNA sequences within feline viral
plaques. J Vet Diag Invest. 2010;22:946–949.
27. Munday JS, Willis KA, Kiupel M, et al. Amplification of three
different papillomaviral DNA sequences from a cat with viral pla-
ques. Vet Dermatol. 2008;19:400–404.
28. Munday JS,WithamAI. Frequent detection of papillomavirusDNA
in clinically normal skin of cats infected and noninfectedwith feline
immunodeficiency virus. Vet Dermatol. 2010;21:307–310.
29. Nespeca G, Grest P, Rosenkrantz WS, et al. Detection of novel
papillomaviruslike sequences in paraffin-embedded specimens
of invasive and in situ squamous cell carcinomas from cats. Am
J Vet Res. 2006;67:2036–2041.
30. O’Neill SH, Newkirk KM, Anis EA, et al. Detection of human
papillomavirus DNA in feline premalignant and invasive squa-
mous cell carcinoma. Vet Dermatol. 2011;22:68–74.
31. Paquette RL, Lee YY, Wilczynski SP, et al. Mutations of p53 and
human papillomavirus infection in cervical carcinoma. Cancer.
1993;72:1272–1280.
32. Park HR, Min SK, Cho HD, et al. Expression profiles of p63, p53,
survivin, and hTERT in skin tumors. J Cutan Pathol. 2004;31:
544–549.
33. Parkin DM. The global health burden of infection-associated can-
cers in the year 2002. Int J Cancer. 2006;118:3030–3044.
34. Parry D, Bates S, Mann DJ, et al. Lack of cyclin D-Cdk com-
plexes in Rb-negative cells correlates with high levels of
p16INK4/MTS1 tumour suppressor gene product. EMBO J.
1995;14:503–511.
35. Scheffner M, Werness BA, Huibregtse JM, et al. The E6 oncopro-
tein encoded by human papillomavirus types 16 and 18 promotes
the degradation of p53. Cell. 1990;63:1129–1136.
36. Smeets SJ, Hesselink AT, Speel EJ, et al. A novel algorithm for
reliable detection of human papillomavirus in paraffin embedded
head and neck cancer specimen. Int J Cancer. 2007;121:
2465–2472.
37. Stricker TP, Kumar V. Neoplasia. In: Kumar V, Abbas AK,
Fausto N, et al. Robins and Cotran Pathologic Basis of Disease.
8th ed. Philadelphia, PA: Elsevier Saunders; 2010:259–330.
38. Sundberg JP, Van Ranst M, Montali R, et al. Feline papillomas
and papillomaviruses. Vet Pathol. 2000;37:1–10.
39. Teifke JP, Lohr CV. Immunohistochemical detection of P53 over-
expression in paraffin wax-embedded squamous cell carcinomas
544 Veterinary Pathology 49(3)
of cattle, horses, cats and dogs. J Comp Pathol. 1996;114:
205–210.
40. Wiest T, Schwarz E, Enders C, et al. Involvement of intact
HPV16 E6/E7 gene expression in head and neck cancers with
unaltered p53 status and perturbed pRb cell cycle control. Onco-
gene. 2002;21:1510–1517.
41. Wilhelm S, Degorce-Rubiales F, Godson D, et al. Clinical, histo-
logical and immunohistochemical study of feline viral plaques
and bowenoid in situ carcinomas. Vet Dermatol. 2006;17:
424–431.
42. Ziegler A, Jonason AS, Leffell DJ, et al. Sunburn and p53 in the
onset of skin cancer. Nature. 1994;372:773–776.
Munday and Aberdein 545