Adenocarcinoma with squamous
metaplasia in the parathyroid gland of a spur-tighed tortoise (Testudo
graeca)
Case report
GÁL János1*, SZABÓ Gyula1, JAKAB Csaba1,
GÉCZY Csaba1, SÁTORHELYI Tamás2
1 Szent István
University, Faculty of Veterinary Medicine, Department of Pathology and
Forensic Veterinary Medicine. István u. 2. H-1078 Budapest.
*E-mail: gal.janos@vipmail.hu
2 Ófalu Pets’ Clinic
Summary
The authors diagnosed
primary adenocarcinoma while dissecting a spur-tighed tortoise carcass.
In the tumour tissue secondary squamous metaplasia was also observed,
as the first reported case in this species. Due to the adenocarcinoma
of the parathyroid gland, the concentration of parathyroid hormone in
the blood increased, which resulted in the extraction of inorganic
matrix substances from the bony tissue of the carapace and plastron. To
identify the tumour type and to characterise its behaviour
immunohistochemical assays routinely used in human diagnostics – such
as Ki-67 and PAN-cytokeratine – were applied by the authors.
Amongst the endocrine
organs of reptiles, parathyroid gland plays an important role in
calcium and phosphorous metabolism. In general, the parathyroid gland
is located near to thyroid gland. In turtles and tortoises the gland is
located between the lobes of the thymus, which makes it difficult to
find it during dissection (4). The gland parenchyma consists only of
main cells with spherical nucleus and smooth granulation in the
cytoplasm. The main cells form bands with one or two cell layers and in
some cases follicular structure can also be observed (1).
The level of the
parathyroid hormone – produced by the parathyroid gland – in the blood
increases by the decrease of calcium level. This causes an increase in
the activity of the osteoclasts in the bony tissue, which results in
the disassembly of the inorganic matrix in the bones. Meanwhile, the
excretion of phosphorous in the kidneys intensifies, and the depletion
of calcium decreases. Parathyroid hormone significantly increases the
intensity of 1,25-dihidrocolecalcipherol synthesis, which stimulates
calcium uptake in the guts (3, 4, 5).
Up to our knowledge, only
adenoma was reported as a parathyroid tumour in reptiles until now. In
lizards, it was detected in green iguanas (Iguana iguana). In tortoises
adenoma was reported in redfoot tortoise (Geochelone carbonaria),
spur-tighed tortoise (Testudo graeca) and desert tortoises
[Xerobates(Gopherus) agassizi]. In each case the parathyroid gland was
swollen in these reptiles. In tortoises the bony tissue of the shell
became soft and could be pressed easily with a finger. In macroscopic
investigations of other organs no pathological malformations were
detected. The pathohistological investigations detected multiple layers
of main cells forming bands. Occasionally, these bands had a spindle
shape and a remarkable eosinophil granulation was observed in the
cytoplasm. In some cases the vacuolization of the cells was also
observed (1, 2, 4). In redfoot tortoises the increased activity of
osteoclasts and the prolification of (collagene fibre) connective
tissue were reported. In the kidneys of tortoises deposition of calcium
salts was also observed (2).
Case
report
On September 5th, 2004, a
carcass of a female spur-tighed tortoise (Testudo graeca) originating
from a private collection was investigated in the Department of
Pathology and Forensic Veterinary Medicine, Faculty of Veterinary
Sciences, Szent István University. The animal was approximately
8 years old, with a carapace length of 15.5 cm.
The tortoise was previously
kept in a home terrarium with a floor space of 100X90 cm. The bedding
of the terrarium was garden soil. The fodder was fresh vegetables and
fruits supplemented with vitamins and mineral salts which were provided
to the animals daily.
Following the external
investigation of the body, the carcass was dissected. Samples were
fixed in formaldehyde solution of 8% from the organs showing
malformations. After embedding the samples in paraffin, sections were
cut and stained with haematoxylin eosin stain.
To study the calcification
status of bones von Kossa silver staining was used. Besides, for
allocating the replicative status of the tumour found, Ki-67 assay and
for detecting the ephitelial marker PAN-cytokeratin immunohistochemical
analysis was also performed. These immunohistochemical reactions were
also performed on the parathyroid gland of a healthy spur-tighed
tortoise, as a control. Amongst the antibodies used, Ki-67 primary
antibody was a DAKO product (Dako, Denmark, Ki-67-MIB 1) while the rest
were mouse monoclonal antibodies produced by Novocastra NCL,
Great-Britain. To detect cytoceratin, the formalin-fixed,
paraffin-embedded tissue samples were reacted with Multi-Cytokeratin
antibody (NCL-AE1/AE3) diluted to 1:50. Besides the blocking of the
endogen peroxidase, protease digestion was performed for 12 minute
durations as a special treatment. For practical use, Ki-67 antibody was
diluted to 1:20. Samples were incubated in citrate buffer for antigen
retrieval at 99 oC, in a microwave oven for 30 minutes, except in the
case of cytokeratin. In each sample, the duration of the antibody
reaction was 30 minutes which was performed at 37 oC in vacuum. As a
second step, it was followed by the avidin-biotin reaction which was
performed at 37 oC in vacuum for 20 and 30 minutes respectively. For
the Ki-67 assay amplification was also performed. For detecting,
aminoethyl carbazol (AEC) was used as a chromogen and slides were
mounted with glycerin-gelatin.
Results
During the dissection of
the tortoise carcass, carapace and plastron were found to be extremely
soft and flexible. The shell was easy to be pressed-in by fingers or to
be slit by a lance; however, no signs of morphological malformations
alluding to previous growth disorders were detected.
After removing the plastron
no pathological abnormality was detected in the inner organs. The
parathyroid glands located in front of the basis of the heart were
enlarged, smoothly contorted. The surface of the glands was scraggly,
the colour was brownish red.
The slit surfaces were
moderately moist and showed a macroscopically homogenous structure.
After staining with haematoxylin eosin, in the sections prepared from
the parathyroid glands, it was clearly visible that the populations of
the polymorphic tumour cells that has a similar appearance as the gland
epithelial cells formed irregular, solid, sometimes slurring bands
unlike the normal nest-like structure. The cells had a large spheric or
oval shaped nucleus with a smoothly granuled chromatine and
perichromasia (Figure 1) and
(Figure 2). The neoplastic cells had prominent
nucleoli. Dividing cells were rarely observed. The irregularly located
proliferating gland epithelial cells had infiltrated the hedging
connective tissue system of the parathyroid gland at several places.
Besides, as a pathological phenomenon islands of squamous metaplasia
were also observed (Figure 3)
in the tumour tissue. The PAN-cytokeratin
immunohistochemical analysis showed a positive result at the metaplasic
areas (Figure 4). The PAN-cytokeratin assay was also performed on the
parathyroid gland of a healthy tortoise, which resulted in a negative
reaction (Figure 5). Dividing ability of tumour cells was found
negative by the performed Ki-67 reaction.
The soft, easily sliceable
bones of the tortoise shell have suggested the decrease, or at some
point the extinction of the inorganic matrix of the bony tissue.
Therefore, for the preparation of sections from the shell,
decalcination of the bony tissue was not necessary. In the haematoxylin
eosin stained embedded samples, the bone trabeculae were almost
completely replaced by collagen fibres (Figure 6). The increased
activity of the osteoclasts was also apparent. After von Kossa silver
staining, only a few small islands of calcium salts were observed in
the bony tissue sections of the test (Figure 7). Virtually the whole
inorganic matrix was completely missing.
In other inner organs no
pathological malformations were detected during macroscopic and
microscopic investigations.
Discussion
Adenocarcinoma, a rare type
of parathyroid tumour with squamous metaplasia was identified causing
softening of the shell of the studied spur-tighed tortoise. After
transforming to tumour cells, the gland epithelial cells typically had
large nuclei and prominent nucleoli. However, these cells did not show
any pronounced aptitude for division. This finding was supported by the
negative outcome of Ki-67 immunohistochemical probe. Due to the high
activity of parathyroid hormone production in the tumorous gland
epithelial cells, the bony tissue of the shell became demineralised
within a short time (primary hyperparathyroidism). The flexible,
rubber-like touch of the dorsal and ventral parts of the shell was
caused by remarkable mobilisation of the mineral elements. The loss of
the inorganic matrix from the shell was already suspected during the
dissection of the animal. This supposition was confirmed by the results
of von Kossa staining of the shell sections, which showed severe
decrease of calcium salts.
The present study is the
first report on squamous metaplasia of the parathyroid gland in
reptiles. The appearance of squamous epithelial cells in the gland
tissue always indicates a pathological condition. These squamous
epithelial cells contain keratohyalin granules in their cytoplasm,
which can be detected by PAN-cytokeratin assay. In the present study
PAN-cytokeratin reaction had a positive result, proving the presence of
squamous epithelial cells in the parathyroid gland. Keratohyalin
granules ought not to be mistaken for the Hassel’s corpuscles of the
thymus.
The tumour tissue detected
in the present study showed no tendency for division, despite the
enlarged nuclei in the cells and the explicit perichromasia. The tumour
formed no metastases. The parathyroid adenocarcinoma consists of tumour
cells with a significant ability to produce hormones. The elevated
parathyroid hormone level caused by such cells activated osteoclast
cells in a relatively short time. The demineralization of the shell is
incidental to the increased activity of the osteoclasts.
The different assays on the
parathyroid gland that are routinely used in human oncology such as
Ki-67 and PAN-cytokeratin were utilizable to characterize certain
features (such as dividing ability, keratohyalin accumulation) of the
cells even in spur-tighed tortoise. Therefore we assume that these
methods are usable to study the evolution of such tumours when
vertebrate species of different evolutionary levels suffer from the
same type of tumour. It is important to note, that the positive
reaction of PAN-cytokeratin assay in a spur-tighed tortoise was very
similar to that of in humans, despite of the phylogenetic distance
between the two species. Our results raise the question, how much the
same types of tumours that develop both in reptiles and in mammals
differ genetically, morphologically and regarding their behaviour, and
show similarities.
Figure 1. Parathyroid gland
with normal structure (control) of a healthy spur-tighed tortoise (T.
graeca)
(back)
Figure 2. Irregularly located gland epithelium cells
with large nucleoli in the parathyroid gland of a spur-tighed tortoise
(T. graeca)
H.-E., 200X
(back)
Figure 3. Squamous metaplasia island in the
parathyroid gland
H.-E., 200X
(back)
Figure 4. PAN-cytokeratin reaction in the cytoplasm
of the cells in squamous metaplasia islands
H.-E., 400X
(back)
Figure 5. PAN-cytokeratin reaction in the
parathyroid gland of a healthy spur-tighed tortoise (T. graeca)
(negative control)
H.-E., 200X
(back)
Figure 6. Transformation in the tissue of the dorsal
part of the shell: connective tissue formation in the place of bony
tissue (osteodystrophia fibrosa). The inorganic matrix is demineralised
H.-E., 40X
(back)
Figure 7. Von Kossa’s staining of a section from the
carapace of a spur-tighed tortoise suffering from parathyroid
adenocarcinom. There is calcium salt only in traces in the shell
H.-E., 40X
(back)