A case of polyostotic osteosarcoma with kidney metastases in a dog

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CASE REPORT
A case of polyostotic osteosarcoma with
kidney metastases in a dog: Histopathology
and microcomputed tomographic analysis
Un cas d’ostéosarcome polyostotique avec metastases
rénales chez le chien : analyse histopathologique et étude en
microtomographie X
E. Aguado a,∗,b, E. Goyenvalle a,b, C. Guintard a
a
Oniris, National Veterinary School of Nantes, UPSP « biologie et biomatériaux du tissus osseux — chirurgie
expérimentale », route de Gâchet, BP 44706, 44307 Nantes cedex 03, France
b
LUNAM université, GEROM—LHEA groupe études remodelage osseux et biomatériaux, Angers, France
KEYWORDS
Osteosarcoma;
Multiple locations;
Metastasis;
MicroCT;
Histopathology
∗
Summary A 7-year- old sexually intact female Leonberg dog was evaluated for chronic lameness of the right forelimb. The bitch showed mild hyperthermia (39.3 ◦ C), a decrease in its
activity, a capricious appetite, a high weight loss (4 kg in 15 days) and a right foreleg lameness.
A careful clinical examination revealed a deformation of the right proximal humerus and right
tibia. Radiographic examination of the right tibia, right humerus showed osteolysis of both cortical and trabecular bone with a periosteal bone proliferation in the vicinal soft tissues. The
owner having refused a bone biopsy, a treatment with NSAIDs and antibiotics was prescribed.
After a marked improvement during the first two weeks, an increase in lameness and activity
was observed. At that time, the owner accepted the bone biopsy. Histopathologic examination
evidenced an osteosarcoma but the amount of available tissue was limited. Due to the poor
prognosis, he declined treatment and decided to euthanize the dog. An osteosarcoma with a
large chondroid component was observed at autopsy together with ossifying kidney metastases.
Histological findings revealed a grade III osteosarcoma. Conventional and undecalcified histology and X-ray microcomputed tomography findings evidenced a large and partially mineralized
osteoid part with a sunburst extension in the soft tissues. This is the first time that microCT
and undecalcified analyses of an osteosarcoma are presented. The osteolytic and metaplastic
bone foci were easily demonstrated by this method.
© 2014 Elsevier Masson SAS. All rights reserved.
Corresponding author.
E-mail address: [email protected] (E. Aguado).
http://dx.doi.org/10.1016/j.morpho.2014.05.002
1286-0115/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Aguado E, et al. A case of polyostotic osteosarcoma with kidney metastases in a dog: Histopathology and microcomputed tomographic analysis. Morphologie (2014),
http://dx.doi.org/10.1016/j.morpho.2014.05.002
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E. Aguado et al.
MOTS CLÉS
Ostéosarcome ;
Localisations
multiples ;
Métastase ;
MicroCT ;
Histopathologie
Résumé Une chienne Leonberg de 7 ans a été évaluée cliniquement pour boiterie chronique
du membre antérieur droit. Elle présentait de plus une légère hyperthermie (39,3 ◦ C), une
diminution d’activité, un appétit capricieux, une perte de poids notable (4 kg en 15 jours). Un
examen clinique attentif a révélé une déformation de l’humérus proximal droit et du tibia
droit. L’examen radiographique de ces deux os a montré une ostéolyse corticale et trabéculaire avec prolifération osseuse périostée en feu d’herbe dans les tissus mous. Le propriétaire
ayant refusé une biopsie osseuse diagnostique, un traitement par AINS et antibiotiques a été
prescrit. Après une nette amélioration au cours des deux premières semaines, la boiterie s’est
majorée et le propriétaire a accepté la biopsie osseuse. L’examen histopathologique objectivait un ostéosarcome mais la quantité de tissu analysable était limitée. En raison du mauvais
pronostic, le traitement a été refusé et la chienne a été euthanasiée. Un ostéosarcome avec une
forte composante chondroïde a été observé à l’autopsie avec des métastases rénales ossifiantes.
Les résultats histologiques ont révélé un ostéosarcome de grade III. Les analyses histologiques
conventionnelles et en technique non décalcifiée, la microtomographie X ont mis en évidence
une forte composante chondroïde, une grande quantité de tissu ostéoïde et une extension de
la tumeur en feu d’herbe dans les tissus mous. C’est la première fois que la microtomographie
X et l’histologie osseuse sans décalcification d’un ostéosarcome sont présentées. L’ostéolyse
et les foyers de métaplasie osseuse ont été facilement démontrés dans un cas d’ostéosarcome
pluricentrique.
© 2014 Elsevier Masson SAS. Tous droits réservés.
Introduction
Osteosarcoma (OSA) is a malignant and rapidly growing
tumor that account for ∼85% of skeletal malignancies in
the dog [2,17]. The tumor is most frequently observed
in middle-aged animals (∼seven years) and almost 90% of
cases occur in dogs weighting more than 20 kg [2]. Largebreed and giant-breed dogs are especially predisposed to
arising of OSA: Labrador retriever, St Bernard, Rottweiler,
Doberman, Golden retriever. . . [15]. The majority of OSA
develops in the appendicular skeleton, at the metaphyseal region of long bones. The foreleg is more frequently
concerned, particularly at the distal radius and proximal
humerus [10,15]. Although a number of epigenetic factors
have been implicated in the development of OSA (fluoride,
ionizing radiations, metal implants, history of bone surgery),
genetic aberrations have been described such as those concerning p53 (tumor suppressor gene) [9,19] or in the CFA34
chromosomal region [14]. OSA of the appendicular skeleton
are highly metastatic tumors to the lung, liver and kidney
[13]. The tumor is also known to be capable to metastasize
to amputation stump or to extend to adjacent bone.
We present here the case of a dog with a plurifocal localization of an OSA associated with kidney
metastasis. Pathological examinations of the different
localizations were done by microcomputed tomography
(microCT), histology on undecalcified and decalcified bone
samples.
Case report
A sexually intact Leonberg dog (female, seven-year-old;
weighting 46.1 kg) was evaluated because of lameness of the
right forelimb. Two months ago, the dog was seen by a general practice veterinarian for this lameness who prescribed
a corticosteroid treatment. Due to the lack of improvement,
the dog was referred to a veterinary specialist in neurology;
no neurological abnormalities were detected. An abdominal
echography did not revealed any abnormality in the liver,
spleen, kidney, bladder and adrenals. One month after this
consultation, the dog was presented to our surgical consultation. The bitch showed mild hyperthermia (39.3 ◦ C), a
decrease in its activity, a capricious appetite, a high weight
loss (4 kg in 15 days) and a right foreleg lameness. A careful clinical examination revealed a deformation of the right
proximal humerus and right tibia. Manipulation of the shoulder was painful. Radiographic examination of the right tibia,
right humerus and thorax was performed and showed osteolysis of both cortical and trabecular bone with a periosteal
bone proliferation in the vicinal soft tissues (sunburst
appearance)(Fig. 1). Thoracic radiographs did not evidence
any tumor metastasis. The hematologic and serum biochemical evaluations revealed an increase in alkaline phosphatase
(1121 UI/L; n = 0—200) and a mild lymphopenia. The diagnosis of bone tumor appeared the most likely. However, due to
the polyostotic lesions (involving both tibia and humerus),
the differential diagnosis of bacterial or fungal osteomyelitis
was considered. The owner having refused a bone biopsy, a
treatment with NSAIDs (firocoxib, Previcox® , Merial, France)
bisphosphonate (tiludronate) and antibiotics (cefalexin,
Rilexine® , Virbac, France) was prescribed. After a marked
improvement during the first two weeks, an increase in
lameness and activity was observed. At that time, the
owner accepted the bone biopsy. It was performed with a
Jamshidi needle (2 mm in diameter) at the tibial crest under
local anaesthesia (diazepam: 0.25 mg/kg, diisopropylphenol: 0.5 mg/kg and morphine 0.1 mg/kg) and isoflurane and
comprized a thicknened cortical bone, soft tissue and bone
marrow.
Histopathologic examination evidenced an osteosarcoma
but the amount of available tissue was limited. Due to the
poor prognosis, he declined treatment and decided to euthanize the dog.
Please cite this article in press as: Aguado E, et al. A case of polyostotic osteosarcoma with kidney metastases in a dog: Histopathology and microcomputed tomographic analysis. Morphologie (2014),
http://dx.doi.org/10.1016/j.morpho.2014.05.002
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Polyostotic osteosarcoma in a dog
3
and humerus shafts (Fig. 1). The tumor had destroyed the
general cortico-trabecular architecture which was replaced
by large amounts of a white fibro-chondroid tissue. Extension in the soft tissues was also evidenced with calcified bone
inside the skeletal muscle fibers. The dissection of the bones
was difficult due to the bone metaplasia that had developed
outside the bone.
No metastasis was observed at the lung, brain, liver and
spleen. A large metastasis was noted in both kidneys which
appeared macroscopically deformed. The kidneys appeared
to contain metastasis with similar characteristics in color
and hardness than the bone tumors. The gross anatomical
findings of the humerus and tibia appear on Fig. 2.
Figure 1 X-ray films of humerus (A) and tibia (B). Note the trabecular bone lysis and periosteal proliferation with a sunburst
appearance in the soft tissues (arrow).
Radiographies de l’humérus (A) et du tibia (B). Notez la lyse
osseuse trabéculaire et la prolifération périostée avec une
apparence en feu d’herbe dans les tissus mous (flèche).
Autopsy findings
At autopsy, the gross macroscopic examination of the bones
revealed very large tumors in the humerus and tibia. The
tumors has developed in the marrow cavity of both tibia
Figure 2 Gross macroscopy aspect of the bone tumor in the
humerus (A) and tibia (B). The tumor has a chondroid aspect
(white arrows) and proliferation of bone inside the soft tissues
is seen (black arrow).
Aspect macroscopique de la tumeur dans l’humérus (A) et le
tibia (B). La tumeur a un aspect chondroïde (flèches blanches)
et la prolifération osseuse à l’intérieur des tissus mous est bien
mise en évidence (flèche noire).
Figure 3 MicroCT of the bone invaded by the tumor. A. Osteolysis observed in the tibial metaphysis. Note that the normal
trabecular microarchitecture has completely disappeared; only
some foci of calcified metaplastic bone are observed. The cortice shows endosteal signs resorption with large eroded areas.
B. Metaplastic bone developed in the soft tissues around the
humeral periosteum. The thin and incompletely calcified bone
trabeculae are responsible for the sunburst appearance noted
on the X-ray images.
MicroCT (microtomographie X) de l’os envahi par la tumeur.
A. Ostéolyse de la métaphyse tibiale. Notez que la microarchitecture trabéculaire normale a complètement disparu et que
des foyers de métaplasie osseuse calcifiée sont observés. La
corticale montre des signes de résorption endostéale avec de
grandes zones érodées. B. Os métaplasique développé dans les
tissus mous autour du périoste de l’humérus. Les néo-travées
osseuses sont minces et incomplètement calcifiées ; elles sont
responsables des lésions en feu d’herbe notées sur les images
radiographiques.
Please cite this article in press as: Aguado E, et al. A case of polyostotic osteosarcoma with kidney metastases in a dog: Histopathology and microcomputed tomographic analysis. Morphologie (2014),
http://dx.doi.org/10.1016/j.morpho.2014.05.002
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Figure 4 Histological aspects of the tumor and its kidney metastases. A. Humeral location: note the multiple chondroid nodules
(arrows) mixed with thin metaplastic trabeculae. B. Higher magnification of the tibial tumor with dystrophic and necrotic chondroid
nodules (arrow) inside a network of metaplastic trabeculae. C. Histochemical identification of sulfated glycosaminoglycans in a
chondroid nodule. D. High magnification of the malignant cells in the left kidney metastasis. Note the morphology of the malignant
osteoblasts at the surface of metaplastic woven bone (high nucleo/cytoplasmic ratio, proeminent nucleoli). Arrow points to a
multinucleated osteoclast. E. Section of the humerus. Note the presence of uncalcified chondroid nodules (c), metaplastic and
calcified bone (m) covered with osteoid tissue (arrow) and areas or necrosis (n). F. Metaplastic bone developing at the surface
of a previous normal trabecula (t); left = bright field, right = polarized light. The ‘‘old’’ trabecula has a lamellar texture and the
metasplasia is made of woven bone. HPS staining after decalcification: A,B, D. Undecalcified sections, toluidine blue: C Goldner’s
stain: E,F.
Aspects histologiques de la tumeur et des métastases rénales. A. Humérus : notez les multiples nodules chondroïdes (flèches)
mélangés avec de minces travées métaplasiques. B. La tumeur tibiale vue à plus fort grossissement avec présence de nodules
chondroïdes dystrophiques et nécrotiques (flèche) à l’intérieur d’un réseau de travées osseuses métaplasiques. C. Identification histochimique des glycosaminoglycanes sulfatés dans un nodule chondroïde. D. Fort grossissement, des cellules malignes
dans la métastase du rein gauche. Notez la morphologie des ostéoblastes malins à la surface de l’os métaplasique fibreux nonlamellaire (rapport nucléo/cytoplasmique élevé, nucléoles proéminents). Les flèches montrent un ostéoclaste multinucléé. E.
Coupe de l’humérus : notez la présence de nodules chondroïdes non calcifiés (c), l’os métaplasique calcifié (m) recouvert de tissu
ostéoïde (flèche) et des zones de nécrose (n). F. Os métaplasique développé à la surface d’une travée normale pré-existante (t) ;
à gauche = éclairage normal, droite = même champ vu en lumière polarisée. La travée « ancienne » a une texture lamellaire et la
métaplasie est faite d’os fibreux non-lamellaire. Coloration HPS après décalcification : A, B, D. Coupes sans décalcification, bleu
de toluidine: C ; trichrome de Goldner : E, F.
Please cite this article in press as: Aguado E, et al. A case of polyostotic osteosarcoma with kidney metastases in a dog: Histopathology and microcomputed tomographic analysis. Morphologie (2014),
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Polyostotic osteosarcoma in a dog
Bone and kidneys were harvested, then fixed and kept in
formalin until analysis in the bone pathology unit.
Microcomputed tomography
Large bone and kidney samples were harvested after having
sectioned the bones on a banding saw. Samples were first
examined by microCT with a Skyscan 1072 X-ray computed
microtomograph (Bruker-microCT, Belgium). Images were
obtained at 80 kV and 100 ␮A with a 1 mm aluminum filter, in
the cone beam mode and a 0.45◦ rotation at each step. The
magnification used for each bone metaphysis was × 15.96;
a pixel corresponding to 19.29 ␮m. The tissue samples were
transferred to radio-lucent plastic vials and analyzed while
in the fixative. For each sample, 2D sections served to
reconstruct 3D models with programs provided by the manufacturer (softwares used: ANT release # 2.05 and Ct-An 2.05
- Skyscan). The reconstructed 3D models were obtained by a
surface-rendering algorithm. The tumor that had developed
within the bone shafts appeared as a radiolucent mass and
could not be imaged. On the other hand, the osteolytic foci
had completely destroyed the trabecular architecture of the
tibia and eroded the endosteal surface. The periosteal metaplasia which had developed inside the soft tissue appeared
as a complex network of very thin trabeculae appended at
the outer cortical surface (Fig. 3).
Histopathology
A sample of each bone and of the kidney was harvested and
included in paraffin after decalcification. Sections were
stained by hematoxylin, phloxin saffron. Glycosaminoglycans were stained by alcian blue and toluidine blue. On
the humerus, a mixed tumor was observed in the center
of the marrow cavity. Dystrophic nodules of malignant
chondrocytes inside an extra cellular matrix rich in sulfated
glycosaminoglycanes was observed (Fig. 4A-C). They were
surrounded by plexiform trabeculae (covered by atypical osteoblasts) mixed with a dense collagenic stroma.
Numerous images of chondrocyte necrosis or ossification
of the chondroid nodules were observed. A high number of
osteoclasts was evidenced at the surface of the metaplastic
bone formations. The cortice appeared lamirated and a
considerable metaplastic bone network extended inside
the soft tissues. In the tibia, very similar images were
encountered and the tumor was composed of half cartilage,
half metaplastic bone.
In the kidney metastases, the normal renal parenchyma
had almost completely disappeared. The metastases were
composed of a dense network of metaplastic trabeculae covered with malignant osteoblasts (Fig. 4D). In all sections
of bone or kidney, the malignant osteoblasts exhibited a
marked anisocytosis, the nuclei had a large central nucleolus
and the mitotic index was elevated. Numerous osteoclasts
were observed in the metaplastic bone, in both the bone
locations and kidney metastases.
Bone and kidney samples were also analyzed undecalcified after embedding in poly(methyl-methacrylate) and
Goldner’s staining [3]. The metaplastic bone had a woven
and non-lamellar texture and the matrix was poorly calcified
(Fig. 4E-F).
5
Discussion
This case report of a OSA in a dog presents several particularities. The osteosarcoma appeared immediately as a
polyostotic tumor with a metaphyseal development both
at the tibial and humerus metaphyses, two classical locations of the tumor at the appendical skeleton [11]. These
rare forms are also encountered in humans [12]. Appendicular OSA have been described to be more aggressive
than axial ones and to have a higher metastatic rate and
higher tumour grade [7]. In our observation, the tumor produced a high amount of chondroid and osteoid tissue in the
bone shafts. It is well known that osteoblasts and chondrocytes have the same stem cell (the CFU-F or stromal
cell) [1]. Local factors such as hypoxia can favour the differentiation in the chondrocytic lineage with activation of
Sox genes [5,16]. Osteoclasts and macrophages have also
been reported to have important roles in osteosarcoma
cells’ dissemination [6]. Otherwise, the stromal cell differentiates into osteoblasts with activation of the runx-2
pathway [8]. So, it is common for OSA to have a mixed
composition. In the soft tissue, extensive development of
woven bone synthesized by malignant cells was observed
and partially mineralized as confirmed by microCT and undecalcified bone histology. The kidney metastases were also
exclusively composed of bone forming cells and elaborated
poorly mineralized trabeculae without cartilage nodules.
According to the classification of Straw et al., this OSA
was a chondro-osteosarcoma, grade III with a high local and
metastatic grade [20]. In a recent study, proximal humerus
and distal femur or proximal tibia location were related with
an increased mortality in the dog [18]. This is the first time
that microCT analysis and undecalcified histology of an OSA
are presented. The technique can be used as a pre-diagnosis
of malignant bone lesions since osteolytic and osteosclerotic
characteristics can be evidenced and are pathognomonic
[4]. In this case, the osteolytic and metaplastic bone foci
were easily demonstrated by this method.
Disclosure of interest
The authors declare that they have no conflicts of interest
concerning this article.
Acknowledgments
This work was made possible by grants from Contrat de plan
état—région ‘‘Pays de la Loire’’ Bioregos2.
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http://dx.doi.org/10.1016/j.morpho.2014.05.002
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Please cite this article in press as: Aguado E, et al. A case of polyostotic osteosarcoma with kidney metastases in a dog: Histopathology and microcomputed tomographic analysis. Morphologie (2014),
http://dx.doi.org/10.1016/j.morpho.2014.05.002