Bull Vet Inst Pulawy 57, 399-405, 2013
DOI: 10.2478/bvip-2013-0069
Expression of p16 (INK4a), cytokeratin 19, and Ki-67
in canine laryngeal squamous cell carcinoma
Małgorzata Kandefer-Gola, Rafał Ciaputa,
Marcin Nowak, Janusz A. Madej
Department of Pathomorphology and Forensic Veterinary Science, Department of Pathology,
Wroclaw University of Environmental and Life Sciences, 50-375 Wroclaw
[email protected]
Received: May 6, 2013
Accepted: September 13, 2013
Abstract
The study aimed at morphological and immunohistochemical characteristics of laryngeal squamous cell carcinomas and
their metastases in canine lymph nodes and lungs. Tissue sections were stained using classical technique with haematoxylin and
eosin. In addition, immunohistochemical studies were performed with p16, cytokeratin, and Ki-67 antibodies. An expression of
all examined antigens was detected in laryngeal tumours, while in tumour metastases only expression of p16 protein and
cytokeratin was demonstrated. The results pointed to higher proliferative potential of the primary tumour than of their metastases.
Key words: dog, laryngeal squamous cell carcinoma, metastases, p16 (INK4a), cytokeratin, Ki-67.
Introduction
Primary laryngeal tumours seldom develop in
companion animals, and if they do, they usually affect
older animals or animals of a moderate age (24, 26).
Most frequently they are observed in dogs and cats (3,
4). Their aetiology remains to be incompletely
clarified; in humans their development is promoted by
smoking of tobacco, excessive alcohol consumption,
exposure to asbestos and nickel, car exhausts,
deficiencies of vitamins A and C, deficiency of iron,
chronic inflammatory conditions, and human papilloma
virus (41). In animals, the studies are continued on
correlation between inhalation of tobacco smoke and
development of tumours in respiratory pathways (7,
24). The larynx may be affected by both benign and
malignant tumours; in dogs development of squamous
cell carcinoma is detected most frequently, while cats
are mostly affected by lymphomas (5, 24, 26, 32).
Individual categories of laryngeal tumours developing
in animals are listed in Table 1 (24).
Squamous cell carcinoma (carcinoma planoepitheliale) represents a tumour, which is the most
frequently encountered in animals of moderate or
advanced age, irrespectively of their gender (37). In
humans, it may be preceded by leukoplakia,
pachyderma, or papillomas in adult individuals (41).
Predisposed dog breeds are boxers, schnauzers, and
white poodles.
Table 1. Categories of laryngeal tumours
Benign
Malignant
rhabdomyoma
squamous cell carcinoma
oncocytoma
adenocarcinoma
leiomyoma
fibrosarcoma
lipoma
osteosarcoma
osteochondroma
chondrosarcoma
rhabdomyosarcoma
lymphoma
mastocytoma
malignant melanoma
The possibility and rate of metastases
development depend on primary tumour location; in
cases of internal organ neoplasms, the metastasis
development is definitely accelerated (38). Upon an
aggressive course of neoplasia, metastases can be noted
in both draining lymph nodes and in distant organs
(36), which rarely occur in humans (41). The neoplasm
is usually a rapidly growing tumour, frequently forming
fine papillae, potentially with erosions or ulcerations on
their surface (14). Under microscope tumour cells
manifest an extensive polymorphism and they infiltrate
the surrounding tissue in the form of nests and streaks
(5). Individual cells contain a round or oval cell nucleus
with centrally located, clearly marked nucleolus and
most frequently an abundant cytoplasm (22).
Depending on the grade of tumour malignancy, a
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variable number of mitotic figures, frequently abnormal
can be noted. Moreover, the tumour structure may
contain infiltrates of inflammatory cells, such as
lymphocytes, histiocytes, neutrophils, plasmocytes, and
macrophages (6, 21). In humans, in addition,
overexpression of EGFR, accumulation of p53,
mutation of TP53 gene, amplification of HER-2, and
loss of 3 p and 9 p, as well as one or a few additional
chromosomes have been detected (41).
The present study aimed at morphological and
immuno-histochemical characteristics of canine
laryngeal squamous cell carcinoma and their metastases
in lymph nodes and lungs.
Material and Methods
The material included five cases of laryngeal
tumours detected upon autopsy. In three cases, the
tumour was accompanied by metastases to regional
submandibular lymph nodes, and in one case
additionally to the lungs. In two cases no metastases
were evident upon macroscopic examination. Tissue
samples were fixed in 7% buffered formalin for 24 h
and processed to form paraffin blocks; 3-4 µm sections
were routinely stained with H&E.
Immunohistochemical examination was performed
in paraffin sections on silanised microscopic slides
(Dako®), dewaxed in xylene, and passed through
alcohol solutions with decreasing concentration, and
finally in water. p16 and cytokeratin antigens were
retrieved in EnVision™ FLEX Target Retrieval
Solution, high pH (50x) (Dako®), and heated in a
water bath at 96ºC for 20 min. Ki-67 antigen was
retrieved in the same manner as p l6 antigen.
Endogenous peroxidase was blocked in EnVision™
FLEX Peroxidase-Blocking Reagent for 10 min.
Subsequently, the sections were overlaid with primary
antibodies: p16 – monoclonal antibody produced in
mouse, Clone DCS-50 (Sigma-Aldrich®) diluted
1:100; cytokeratin (CK) - monoclonal human antibody
produced in mouse, Clone RCK108 (Dako®) diluted
1:50; Ki-67 - human antibody produced in mouse,
Clone MIB-1 (Dako®) diluted 1:50. Negative and
positive controls were included. As positive control,
normal, non-tumour tissue treated with the same
antibody reagents like in neoplastic tissue, was used. In
negative control, antibodies were omitted.
Expression of Ki-67 was graded using a semiquantitative scale for evaluation of percentage of
positive cells (0–5% - no reaction (-), 6%–25% scanty
reaction (+), 26%–50%, moderate reaction (++), >50%
intense reaction (+++). Upon evaluation of p16 and
cytokeratin, the scale of Remmele (10, 40) was used,
the technique taking into account both percentage of
positive cells (A) and intensity of reaction colour (B),
while the final score represented a score of the two
parameters, ranging between 0 and 12 points (no
reaction – 0 pts (-); scanty reaction 1-2 pts (+),
moderate reaction 3-4 pts (++), intense reaction 6-12
pts (+++) (Table 2).
Table 2. Scale according to Remmele
A
0 pts – absence of cells with
positive reaction
1 pts – up to 10% positive cells
2 pts – 11%–50% positive cells
3 pts – 51%–80% positive cells
4 pts – > 80% positive cells
B
0 pts – no reaction colour
1 pt – weak reaction colour
2 pts – moderately intense
reaction colour
3 pts – intense reaction colour
Microphotographs of all the examined neoplastic
lesions were taken using Olympus BX53 optical
microscope (Olympus, Japan), coupled with ColorView
IIIu digital camera (Olympus, Japan).
Results
In all examined cases, laryngeal tumours formed a
diffuse, irregular, grey-pink proliferative lesions,
embracing laryngeal surface (Fig. 1).
Regional lymph nodes were intact or slightly
enlarged due to a secondary inflammatory process or,
in the cases of metastases (three cases), significantly
enlarged, cavernous, with obliterated structure (Fig. 2).
In the case of sample with pulmonary metastases
(a single case) cross-section of lobes displayed variable
size, cream-pinkish nodules of a uniform cross-section
(Fig. 3).
Histopathological examination of laryngeal
samples
demonstrated
neoplastic proliferation,
classified according to WHO recommendations as
squamous cell carcinoma, infiltrating the region of
laryngeal cartilages (Fig. 4). Neoplastic cells, arranged
in nests separated by strands of connective tissue,
manifested a significant polymorphism. Cells with
large nuclei, with clearly marked nucleoli and visible
figures of mitotic division, prevailed. Haemorrhages
with subsequent accumulation of haemosiderin
granules were also noted. Moreover, an infiltration of
inflammatory cells, composed predominantly of
lymphocytes, and foci of necrosis were also present.
Sections of neoplastically altered lymph nodes
manifested diffuse regions of necrosis, lymphocytic
inflammatory foci, and diffuse infiltrates of neoplastic
cells (Fig. 5).
In samples of pulmonary tissue, well delineated
from pulmonary tissue, focal metastases were observed,
presenting neoplastic structure of squamous cell
carcinoma type. Moreover, the surrounding tissue
contained slight amounts of oedematous fluid,
accompanied by haemostasis, haemorrhages, and
granules of hae-mosiderin (Fig. 6).
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401
Fig. 1. Neoplastic infiltration of the larynx
Fig. 5. Histological pattern of lymph node metastasis resulting from
laryngeal squamous cell carcinoma. H&E, 100x
Fig. 2. Cross-section of an affected submandibular lymph node
Fig. 6. Metastatic focus in lungs, haemostasis, and oedema in the
surrounding pulmonary tissue. H&E, 100x
Fig. 3. Metastatic lesions in lungs
Fig. 7. Expression of Ki-67 in primary tumour. Immunostaining for
Ki-67, 200x
Fig. 4. Infiltration of squamous cell carcinoma cells in laryngeal
cartilage. H&E, 40x
Expression of neoplastic markers in individual
organs was detailed in Table 3. In analysis of the
intensity of Ki-67 protein expression, a relatively
pronounced nuclear reaction at the level of (++) was
detected in the primary tumour (Fig. 7), while in
metastases expression of the marker was lower.
In tests using p16-specific antibodies a very strong
(+++, 8 in Remmele’s scale) cytoplasmic reaction was
detected in all neoplastic lesions in larynx, lymph
nodes and lungs (Fig. 8).
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Discussion
Fig. 8. Strong expression of p16 in neoplastic tissue. Immunostaining
for p16, 100x
Fig. 9. Pronounced expression of cytokeratin in laryngeal tumour.
Immunostaining for cytokeratin, 100x
Upon evaluation of cytokeratin expression also a
very pronounced cytoplasmic and membranous
reaction (+++, 9 in Remmele’s scale) was disclosed
both in the laryngeal tumour and in sections of
metastatic tumours (Fig. 9).
Table 3. Expression of tumour markers in primary tumour and in
metastases
Marker
Case number
Laryngeal
tumour
Lymph node
Lungs
Ki-67
1
+++
++
+
2
++
+
3
++
+
4
++
5
+
1
+++
+++
2
+++
++
3
+++
+++
4
+++
5
++
1
+++
+++
2
+++
+++
3
+++
+++
4
+++
5
+++
p16
cytokeratin
+++
+++
In animals laryngeal tumours develop infrequently
and they are estimated to be detected in around 0.02%
of all bioptic and histopathological studies (9). Most of
the studies published until now have involved
individual cases of affected animals. Squamous cell
carcinoma represents one of the most frequent dermal
tumours of epithelial origin, while in internal organs it
is relatively rare (42). In most cases it develops in
tonsils, stomach, and urinary bladder (38). Cases of
squamous cell carcinoma in the larynx were described
in dogs, cats, horses, and the black bear (16, 26, 35).
The syndrome of non-specific signs, such as coughing,
aphonia, lack of appetite, or apathy, frequently
bypassing the accessory studies during the diagnostic
process, cause difficulties in establishing the correct
diagnosis. In the studies diffuse, massive lesions
involving the entire laryngeal surface have been
observed. This may point to highly aggressive nature of
the neoplastic tumour, and to difficulties in diagnosis
and treatment at early stages of the tumour
development.
As mentioned above, the rate of metastasis
development depends on primary location of the
tumour (38). While dealing with dermal squamous cell
carcinoma, it should be remembered that the lesion
expresses local invasiveness and metastatic potential
primarily to local lymph nodes. It is not until, the
disease process is advanced that secondary neoplastic
lesions can be detected in more distant organs. In the
case of squamous cell carcinoma of inner organs,
including larynx, the course of neoplastic process is
definitely more aggressive. The tumour manifests an
intense growth, a pronounced invasive character, and a
high rate of metastasis development in surrounding
tissues, local lymph nodes, and the more distant organs
(22). In the study, metastatic lesions have been detected
in both local lymph nodes and lungs. All examined
tumours demonstrated infiltration of laryngeal cartilage
and high mitotic index. Furthermore, no significant
differences in the immunoexpression level of primary
tumour and metastases were observed. This confirmed
our assumptions
concerning
a highly invasive
character of the neoplastic process in all cases.
At present, immmunohistochemical examination
becomes an indispensable element of histopathological
investigations. Using various markers, it is possible to
confirm the diagnosis by verifying the efficacy of the
conducted treatment or to define prognosis. In
veterinary medicine, such tests are not routinely
performed, mainly due to high costs of the antibodies
and equipment required for the immunohistochemical
laboratory. An additional problem involves application
of antibodies specific for human tissues and the
relatively insufficient available and verified data on
cross-reactions with animal tissues. In the
investigations, we have applied antibodies used in
diagnosis of laryngeal tumours in humans, i.e.
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antibodies specific for p16, cytokeratin, and Ki-67.
Their reactivity with canine tissues has been confirmed
in earlier studies (18, 25, 27–30).
In the process of establishing the diagnosis, the
first stage involves determination of origin of
neoplastic tumour. In unclear situations, expression of
some markers, including cytokeratins may be helpful.
Cytokeratins (CK) represent one of the most important
elements of cytoskeleton in epithelial cells of humans
and animals. They belong to the group of cytoplasmic
proteins forming i.e. intermediate filaments (8, 19, 43).
The proteins may be categorised into the soft and solid
forms. In the group of cytokeratins, two subfamilies
differing in molecular weight, including cytokeratins I
(CK9-CK20) and cytokeratins II (CK1-CK8), can be
distinguished (23). Cytokeratins undergo the process of
ubiquitination (taking place during proteasomemediated protein degradation), which results in
modification of structure and, indirectly, function of
various proteins. Deformed cytokeratins disrupt shape
of cytokeratin network in the cytoplasm, thereby its
structure becomes more loose and the natural
cytoskeleton
structure
becomes
destroyed.
Accumulation of the altered proteins increases
aggressiveness and invasiveness character of the cells.
Furthermore, processes of mitosis and apoptosis
become disturbed, which promotes immortality of the
altered cells (15). Expression of cytokeratins is used,
first of all, for distinguishing neoplastic cells of
epithelial origin from undifferentiated or poorly
differentiated neoplastic cells stemming from the other
germ layers (17). Moreover, cytokeratins were applied
in evaluation of the degree to which a tissue is
abnormally differentiated (11). Cytokeratin 19 was
used in the study due to its versatility and prognostic
value in patients with squamous cell carcinoma. In the
performed studies on laryngeal carcinomas, a
pronounced (+++) cytokeratin expression has been
detected in all cases. Positive result of such staining
allowed to confirm epithelial origin of primary tumours
and metastases.
In the diagnostic process, early detection and
diagnosis of a tumour are of a primary significance.
The biomarker p16 (INK4a) allows to detect
disturbances in the cell cycle, pointing to an abnormal
process of cell division. The protein plays a particular
role in the negative control of the cell cycle. It
represents an inhibitor of cyclin-dependent kinases
(CDKI), which decreases the rate of cell cycles through
inactivation of specific CDKI, such as kinases of D1,
CDK4, and CDK6 type (40). Therefore, CDKs
inhibitors are considered to form one of the signal
cascade elements, which inhibit or slow down
replication in the course of cell differentiation process
(31). Inactivation of p16 was noted in neoplastic cell
lines, which may suggest its antineoplastic effect (33).
On the other hand, several authors have demonstrated
high expression of the protein in malignant tumours,
such as uterine carcinoma and adenocarcinoma,
403
lymphatic leukaemia, malignant melanoma, and
squamous cell carcinoma (1, 39, 44). Moreover, the
expression was found to be correlated with the grade of
tumour malignancy (1). Effectiveness of the marker in
description of behaviour manifested by other tumours
(malignant melanoma or gastric carcinoma) found
confirmation in earlier studies (18, 25, 34). Until now,
no investigations have been performed using p16
(INK4a) protein in lesions of laryngeal squamous cell
carcinoma type in dogs. Summing up, positive reaction
for p16 (INK4a) provides an information on
disturbances in the cell cycle, and, indirectly, on
neoplastic tumour growth rate and potential for
development of metastases (10). In the study, high
levels of expression of p16 (INK4a) protein were
detected in all examined laryngeal carcinomas and their
metastases. As mentioned earlier, this points to
disturbances in cell cycle and, thus, to the rate of
cellular divisions and growth of the tumours.
Furthermore, in one case the observed expression level
was higher in primary tumour than in metastasis.
In histological evaluation the subsequent
important element involves assessment of tumour
proliferative potential. The rate of cell proliferation
may be estimated through expression of various
proteins, including Ki-67. Its presence can be detected
in all phases of cell cycle except of G0 phase. Starting
at G1 phase of the cycle, concentration of the protein
continues to grow, to reach its maximum level at M
phase (20). Expression of the protein is best visible in
active cells, ready for proliferation (2). In addition, in
humans a positive correlation was detected between
cells manifesting expression of Ki-67 and rate of
primary tumour growth, and potential to manifest a
relapse (12). The correlation was confirmed also in
veterinary medicine (27–30). Following analysis of 60
mammary gland tumours in bitches, Gizinski et al. (13)
documented
relationship
between
pronounced
expression of Ki-67 protein and low level of
differentiation of the tumour cells, and presence of
metastases. In the studies, using Ki-67 proliferative
antigen, its moderate expression (++) was documented
in the primary tumour and its low expression in
metastases. This indicates a higher proliferative
potential of cells in primary tumour, its dynamic
growth, which correlated with a poor prognosis for the
patient.
Summing up, it may be stated that
histopathological analysis and immunohistochemical
tests with an appropriately selected panel of antibodies
allow for a correct and rapid diagnosis of neoplasms. In
several cases they may also enable the clinician to
define prognosis, and to select an effective therapy of
the defined type of tumour.
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