In vitro effects of the activity of novel platinum (II) complex in canine and human cell lines

The anticancer activity of novel platinum derivative, a complex of platinum with tris(2‐carboxyethyl)phosphine (Pt‐TCEP), has been evaluated in canine (D‐17) and human osteosarcoma (U2‐OS) cell lines. Viability of cells after incubation for 24 or 72 hours with increasing concentrations (0.625, 1.25, 2.50, 5, 10 and 20 μM) of Pt‐TCEP was tested in an MTT assay and compared to effect of cisplatin. Longer‐term effect of Pt‐TCEP was evaluated in the colony‐forming unit assay after 24 hours exposure to the Pt‐TCEP (2 and 3 μM) and subsequent incubation for 2 weeks. The influence of the compound on the cell cycle was measured after 24 hours treatment with Pt‐TCEP (3 μM). Its pro‐apoptotic activity was examined after 24 hours treatment with Pt‐TCEP (1.25, 2.50, 5, 10 and 20 μM) using flow cytometry. Expression of main proteins involved in apoptosis was measured after exposure for 24 hours to 3 or 5 μM Pt‐TCEP in Western Blot. The compound much more effectively decreased cell viability than cisplatin in case of both cell lines. IC₅₀ of Pt‐TCEP was 5.93 ± 0.12 in D‐17 and 3.45 ± 0.14 in U2‐OS cell lines after 24 hours, and 1.77 ± 0.14 in D‐17 and 1.53 ± 0.11 in U2‐OS after 72 hours (P < .05). The compound arrested cells in the G2/M phase and inhibited the ability of cells to form colonies. Pt‐TCEP induced caspase‐dependent apoptosis. The expression of the anti‐apoptotic Bcl‐X_L protein was decreased after Pt‐TCEP treatment in both cell lines. The results confirmed anti‐cancer activity of Pt‐TCEP against canine and human osteosarcoma cell lines.     

Malignant mesenchymoma with widespread metastasis including bone marrow involvement in a dog

A male castrated Golden Retriever was presented for evaluation of a large mass over the left shoulder extending to the lower part of the neck that had been present for an extended period of time, but had a recent history of rapid growth. Previous aspirates of the mass were consistent with a lipoma. The mass was surgically excised and was diagnosed as an extraskeletal osteosarcoma based on histopathology. After surgery, the dog was initiated on a chemotherapy protocol with carboplatin and metronomic cyclophosphamide. He became neutropenic, anemic, and thrombocytopenic 14 days after the carboplatin treatment was administered. The neutropenia resolved, but the anemia and thrombocytopenia progressed. A bone marrow aspirate revealed erythroid hypoplasia, myeloid hyperplasia with a predominance of early precursors, and a subset of cells that made up 20% of the total population that were reported as bizarre and unclassifiable. These cells were discrete in nature and were thought to be hematopoietic in origin. The dog was euthanized due to deterioration of the clinical condition. On postmortem examination, widespread metastasis involving the lungs, liver, kidney, heart, and bone marrow was found. Histopathology of the tumor lesions determined 2 distinct malignant populations of liposarcoma and osteosarcoma, consistent with malignant mesenchymoma. However, the possibility of 2 separate neoplastic processes cannot be definitively excluded. This is the first report of bone marrow metastasis of a malignant mesenchymoma in a dog.     

Osteosarcoma in the proximal humerus of a mare

A 15‐year‐old Quarter Horse mare was examined for significant and progressive lameness in the right front limb. On physical examination, muscle atrophy over the scapular and pectoral regions were noted. A pain response was elicited on palpation of the scapulohumeral joint. No other abnormalities were noted on examination of the limb. Diagnostic nerve blocks ruled out a source of lameness in the distal limb. Arthrocentesis of the scapulohumeral joint was performed and results were within normal limits. Radiographs of the right scapulohumeral joint revealed an extensive, aggressive bone lesion in the proximal humerus, suspected to be an osteosarcoma. After a course of nonsteroidal anti‐inflammatory therapy, minimal improvement to the lameness was noted. The owner elected to subject the horse to euthanasia due to the lack of significant response to treatment. Post mortem radiographic and histological examinations determined a definitive diagnosis of an osteosarcoma in the proximal humerus.     

Osteosarcoma in the femur of a horse

A 27-year-old Arabian pony gelding presented for evaluation of weight loss, intermittent sheath oedema, persistent neutropenia and thrombocytopenia, and acute left hindlimb swelling and gait abnormality. Clinical findings included swelling, heat and sensitivity localised over the left greater trochanter, mild to moderate ventral and sheath oedema, a left hindlimb post-legged gait, and off-loading of weight from the left hindlimb at rest. Initially, neutropenia and thrombocytopenia were confirmed on bloodwork, but neutrophilia and thrombocytopenia persisted as the case progressed. Diagnostic imaging (radiography and percutaneous ultrasonography) of the left hindquarters revealed an aggressive, mixed proliferative and lytic bony lesion of the proximal left femur as well as associated muscle fibre disruption and a soft tissue mass. A percutaneous core biopsy of the lesion led to the diagnosis of osteoblastic osteosarcoma on histopathology. Pain and inflammation associated with the lesion was medically managed in the hospital with mild improvement. Additionally, palliative radiation was performed under general anaesthesia. Unfortunately, before a response to palliative radiation could be assessed, the patient was subjected to euthanasia due to development of acute neurological signs.     

ACCURACY OF MAGNETIC RESONANCE IMAGING FOR ESTIMATING INTRAMEDULLARY OSTEOSARCOMA EXTENT IN PRE-OPERATIVE PLANNING OF CANINE LIMB-SALVAGE PROCEDURES

The objective of this work was to compare the accuracy of radiographs and magnetic resonance imaging (MRI) for estimating appendicular osteosarcoma margins. The accuracy of computed tomography (CT) and bone scintigraphy was also assessed when these studies were available. Eight dogs with appendicular osteosarcoma underwent radiographic and MRI of affected limbs. In addition, bone scintigraphy was performed in six dogs and CT examination was performed in five dogs. Two observers jointly measured tumor length on all imaging studies. Correlative gross and histologic evaluation of all affected limbs was performed to determine tumor extent as measured from the nearest articular surface. Results from imaging studies were compared to gross and microscopic morphometry findings to determine the accuracy of each modality for determining tumor boundaries. MRI images were accurate with a mean overestimation of actual tumor length of 3 +/- 13%. T1-weighted non-contrast images were superior in identifying intramedullary tumor margins in most instances whereas contrast-enhanced images provided supplemental information in two dogs. Lateromedial and craniocaudal radiographs overestimated tumor length by 17 +/- 28% and 4 +/- 26%, respectively. Scintigraphy and CT overestimated tumor margins by 14 +/- 28% and 27 +/- 36%, respectively. MRI appears to be an accurate diagnostic imaging modality in determining intramedullary osteosarcoma boundaries. MRI should be considered as part of a pre-operative assessment of appendicular osteosarcoma, particularly when a limb-sparing procedure is contemplated.     

Immune dysregulation and osteosarcoma: Staphylococcus aureus downregulates TGF‐β and heightens the inflammatory signature in human and canine macrophages suppressed by osteosarcoma

Since William Coley utilized bacterial immunotherapy to treat sarcomas in the late 19th century, an association between infection and improved survival has been reported for human and canine osteosarcoma patients. One of the reasons for this improved survival is likely a reactivation of the host immune system towards an inflammatory anti‐tumour response, and one of the key players is the macrophage. Yet, despite their importance, the response of macrophages to infectious agents in the context of osteosarcoma has not been thoroughly evaluated. The aim of this study was to evaluate how in vitro exposure to a bacterial agent (Staphylococcus aureus) influenced canine and human macrophage differentiation in the presence of osteosarcoma. Our hypothesis was that S. aureus would, in the presence of osteosarcoma, induce a macrophage phenotype with significantly increased inflammatory signatures. Consistent with our hypothesis, human macrophages co‐cultured with osteosarcoma and S. aureus exhibited increased IFN‐γ, TNF‐α and IL‐12p70 cytokine secretion, decreased TGF‐β cytokine secretion and increased mRNA expression of TNF‐α when compared with macrophages co‐cultured with osteosarcoma and to macrophages cultured alone. Canine macrophages similarly exhibited increased IFN‐γ and TNF‐α cytokine secretion, decreased TGF‐β cytokine secretion, increased mRNA expression of TNF‐α and increased surface receptor expression of CD80 when co‐cultured with osteosarcoma and S. aureus. Collectively, the findings of this study suggest that infection upregulates the inflammatory immune response to counteract osteosarcoma‐induced immune suppression. This work informs a potential therapeutic strategy to optimize inflammatory stimuli for triggering an anti‐osteosarcoma macrophage response.     

Spontaneous Extraskeletal Osteosarcoma in a Rabbit (Oryctolagus cuniculus): Histopathological and Immunohistochemical Findings

A spontaneously occurring subcutaneous mass in the left forelimb of a nine-year-old rabbit (Oryctolagus cuniculus) was examined histopathologically and immunohistochemically. Clinically, edema and hemorrhage were seen around the mass. No connection of the tumor mass to the appendicular skeleton was found. The tumor was arranged in a solid growth pattern and irregular bundles, and neoplastic cells were polygonal to spindle-shape. Osteoid (positive for osteocalcin) and multinucleated giant cells were diffusely or focally seen. Neoplastic cells were positive for vimentin, osterix and Ki-67, indicating the nature of osteoblasts with proliferating activity, but negative for α-smooth muscle actin, desmin or CD204. Based on these findings, a diagnosis of extraskeletal osteosarcoma was made, a very rare tumor both in laboratory and pet rabbits.     

Chondroblastic Osteosarcoma in Two Related Spiny-Tailed Monitor Lizards (Varanus acanthurus)

Two male spiny-tailed monitor lizard (Varanus acanthurus) broodmates were diagnosed with chondroblastic osteosarcomas associated with the pelvic girdle. One lizard was 6 months old and the other 5 months old at the time of diagnosis. Grossly, the tumors appeared as large, firm, multilobulated masses that were white on cut surface and firmly adhered to the underlying bone. Histologically, both tumors were characterized by proliferation of neoplastic spindloid to stellate cells that encompassed the vertebrae and invaded into the vertebral and pelvic bones. Both osteoid production and multifocal chondrous differentiation were observed within neoplastic masses. Retinoblastoma 1 was absent in neoplastic cells. Even though osseous neoplasms are rare amongst reptiles, Varanus spp. may have a genetic predilection for developing chondroblastic osteosarcoma.     

CASE REPORT: Intraocular osteosarcoma in an Umbrella Cockatoo (Cacatua alba)

An adult, presumed intact female Umbrella Cockatoo (Cacatua alba), presented with acute hemorrhage from an intraocular mass that perforated through the right cornea. Computed tomography scanning revealed a large soft tissue mass in the right orbit, invading and displacing the globe laterally, and destroying the scleral ossicles. There was no evidence of bony changes of the orbit or extension of the mass into the optic nerve or brain. Exenteration and mass removal were performed, and osteosarcoma was diagnosed via histopathology. Radiotherapy was delivered with an orthovoltage unit to a total dose of 68 Gray delivered in 17 fractions over 6 weeks. The bird recovered well from treatment, but died 2 months after the last radiation session with neurological signs. Necropsy was not performed. To our knowledge, this is the first case of an intraocular osteosarcoma reported in a bird, and the first case of attempted treatment of osteosarcoma in a bird by a combination of surgery and radiation therapy.     

Spontaneous extraskeletal osteosarcoma in the stomach of an aged f344 rat

Extraskeletal osteosarcoma is a very rare tumor in humans and animals including rats. This paper describes a case of extraskeletal osteosarcoma observed in the glandular stomach of an aged female Fischer 344 rat. Grossly, a whitish solid mass was observed at the greater curvature of the glandular stomach. Histologically, the tumor consisted of both atypical polygonal and pleomorphic spindle-shaped cells, with pleomorphic nuclei, and it contained variable amounts of osteoids and small clumps of mature bone tissue. In addition, mitotic figures were frequently observed. Neither invasion of the muscle layer or vessels in the stomach nor metastasis to distant organs was detected. There were no skeletal tumors in the body. Immunohistochemically, the tumor cells were positive for osteocalcin, osteonectin, vimentin and S-100 protein. Judging from these results, the present tumor was diagnosed as extraskeletal osteosarcoma. This is the first report of spontaneous extraskeletal osteosarcoma arising from the stomach in a rat.