Successful surgical resection of a multilobular osteochondrosarcoma arising from the costal cartilage in a cat.

A 10-year-old neutered male cross-bred cat was referred to our clinic for a solid mass tightly fixed to the right side of the thoracic wall from the 2nd to 4th ribs. Computed tomography revealed the mass had remarkable calcifications and arose from the 3rd costal cartilage. After removal, it was diagnosed histopathologically as a multilobular osteochondrosarcoma (MLO). For tumor resection, extremely wide surgical margins included 6 costal cartilages and 3 sternal segments were required; however, the tumor was successfully resected, followed by reconstruction of the thoracic wall using artificial materials. The cat recovered uneventfully and was good in health for ~4 y. This is apparently the first report of surgical resection of MLO from the costal cartilage of a cat. Key clinical message: To our knowledge, this is the first report of MLO from the costal cartilage in a cat, demonstrating aggressive surgical resection despite extremely wide surgical margins.

Résection chirurgicale réussie d'un ostéochondrosarcome multilobulaire provenant du cartilage costal chez un chatUn chat croisé mâle castré de 10 ans a été référé à notre clinique pour une masse solide bien fixée sur le côté droit de la paroi thoracique de la 2e à la 4e côte. La tomodensitométrie a révélé que la masse présentait des calcifications remarquables et provenait du 3e cartilage costal. Après retrait, il a été diagnostiqué histopathologiquement comme un ostéochondrosarcome multilobulaire (MLO). Pour la résection tumorale, des marges chirurgicales extrêmement larges comprenaient 6 cartilages costaux et 3 segments sternaux; cependant, la tumeur a été réséquée avec succès, suivie d'une reconstruction de la paroi thoracique à l'aide de matériaux artificiels. Le chat s'est rétabli sans incident et était en bonne santé pendant environ 4 ans. Il s'agit apparemment du premier rapport de résection chirurgicale de MLO du cartilage costal d'un chat.Message clinique clé:À notre connaissance, il s'agit du premier rapport de MLO du cartilage costal chez un chat, démontrant une résection chirurgicale agressive malgré des marges chirurgicales extrêmement larges.(Traduit par Dr Serge Messier).

Use of a haired angularis oris axial pattern flap in a dog to correct a large oronasal fistula secondary to resection of a hard palate multilobular osteochondrosarcoma.

A 6-year-old neutered male mixed-breed dog underwent curative-intent surgical resection of a hard palatal multilobular osteochondrosarcoma and closure of the defect using bilateral buccal mucosal flaps. However, failure of the flaps resulted in a massive hard palatal defect that was subsequently repaired using a haired skin angularis oris axial pattern flap. This report describes the clinical outcome using this surgical approach and novel complications encountered. Key clinical message: The haired skin angularis oris axial pattern flap appears to be a suitable and robust option for reconstruction of large palatal defects.

Utilisation d'un lambeau cutanée poilus avec rotation axiale au niveau de l'artère angularis oris chez un chien pour corriger une fistule oronasale volumineuse secondaire à la résection d'un ostéochondrosarcome multilobulaire du palais dur. Un chien croisé mâle castré de 6 ans a subi une résection chirurgicale à visée curative d'un ostéochondrosarcome multilobulaire du palais dur et une fermeture de l'anomalie par des lambeaux de la muqueuse buccale. Cependant, la défaillance des lambeaux a entraîné un défaut important du palais dur qui a ensuite été réparé à l'aide d'un lambeau de peau avec poils avec rotation axiale au niveau de l'artère angularis oris. Ce rapport décrit les résultats cliniques de cette approche chirurgicale et les nouvelles complications rencontrées.Message clinique clé :L'utilisation d'un lambeau de peau avec poils avec rotation axiale au niveau de l'artère angularis oris semble être une option appropriée et robuste pour la reconstruction des défauts importants du palais.(Traduit par Dr Serge Messier).

Three cases of dogs with osteosarcoma of the forelimb treated with liquid nitrogen for limb-sparing surgery using autologous bone.

Osteosarcoma treatment with limb-sparing surgery using liquid nitrogen can be applied to canine patients experiencing diminished quality of life after leg amputation. In particular, forelimb amputation may affect gait more than hindlimb amputation. In this study, limb-sparing surgery using liquid nitrogen was applied to primary osteosarcomas arising in the proximal scapula of a Welsh Corgi, the proximal humerus of a Golden Retriever, and the distal radius of a Great Pyrenees, according to the protocol of Tsuchiya et al. In all cases, postoperative radiographic examination revealed bone union between the treated and matrix bones. All patients recovered their gait postoperatively. These results suggest that limb-sparing surgery using liquid nitrogen-treated autologous bone is an effective option for patients with osteosarcoma.

Prospective pilot study utilizing changes in quantitative values obtained on serial fluorine-18 fluorodeoxyglucose (18F-FDG) positron emission tomography-computed tomography (PET/CT) in dogs with appendicular osteosarcoma before and after stereotactic body radiation therapy (SBRT) and carboplatin chemotherapy to assess for prediction of survival and therapeutic effectiveness.

Serial fluorine 18 fluorodeoxyglucose (18F-FDG) positron emission tomography-CT (PET/CT) is commonly used in human oncology to prognosticate and evaluate for therapeutic effectiveness. In this pilot study, dogs with naturally occurring appendicular osteosarcoma were evaluated with serial 18F-FDG PET/CT in an attempt to assess for response to therapy, prognostic factors, and appropriateness of imaging intervals. Fourteen dogs were enrolled in the trial. All dogs had the initial 18F-FDG PET/CT (PET1), with nine dogs having their end-of-therapy 18F-FDG PET/CT (EoT PET) 3 months after stereotactic body radiation therapy (SBRT) to the primary tumor. The median percent change from the PET1 to the EoT PET for the standard uptake value maximum (SUVmax%) was -58% (range: -17 to -88%), metabolic tumor volume (MTV%) was -99.8% (range: -65 to -100%), and total lesion glycolysis (TLG%) was -99.8% (range: -75 to -100%), all of which were significant (P < .05, <.05, and <.05, respectively). On evaluation, it was found that volumes of GTV and CTV were significant for survival (P < .05 and <.05), MTV1, TLG1, and SUVmax on the EoT PET (SUVmaxEoT) were predictive of metastasis (P < .05), and the SUVmax% was significantly correlated to the time to first event (P < .05). Based on this data, serial 18F-FDG PET/CT performed 3 months after SBRT can show a significant reduction in avidity, and the quantitative data collected may help predict metastatic disease in canine appendicular osteosarcoma.

Metastatic Osteosarcoma of the Distal Femur in a Korean Water Deer (Hydropotes inermis argyropus).

A free-living female Korean water deer (Hydropotes inermis argyropus) was found with swelling in the left femur. Radiographic and histopathologic examination confirmed distal femoral osteosarcoma with metastases to the inguinal lymph node and the lungs; there are no previous reports of osteosarcoma in water deer.

Single-cell RNA sequencing reveals the cellular and molecular heterogeneity of treatment-naïve primary osteosarcoma in dogs.

Osteosarcoma (OS) is a heterogeneous, aggressive malignancy of the bone that disproportionally affects children and adolescents. Therapeutic interventions for OS are limited, which is in part due to the complex tumor microenvironment (TME). As such, we used single-cell RNA sequencing (scRNA-seq) to describe the cellular and molecular composition of the TME in 6 treatment-naïve dogs with spontaneously occurring primary OS. Through analysis of 35,310 cells, we identified 41 transcriptomically distinct cell types including the characterization of follicular helper T cells, mature regulatory dendritic cells (mregDCs), and 8 tumor-associated macrophage (TAM) populations. Cell-cell interaction analysis predicted that mregDCs and TAMs play key roles in modulating T cell mediated immunity. Furthermore, we completed cross-species cell type gene signature homology analysis and found a high degree of similarity between human and canine OS. The data presented here act as a roadmap of canine OS which can be applied to advance translational immuno-oncology research.

Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer.

BACKGROUND: Natural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers in the context of an intact immune system, representing a valid cancer immunotherapy model. Previously, CD5 depletion of peripheral blood mononuclear cells (PBMCs) was used in dogs to isolate a CD5dim-expressing NK subset prior to co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. This study aimed to assess NK activation, expansion, and preliminary clinical activity in first-in-dog clinical trials using a novel system with unmanipulated PBMCs to generate our NK cell product. METHODS: Starting populations of CD5-depleted cells and PBMCs from healthy beagle donors were co-cultured for 14 days, phenotype, cytotoxicity, and cytokine secretion were measured, and samples were sequenced using the 3'-Tag-RNA-Seq protocol. Co-cultured human PBMCs and NK-isolated cells were also sequenced for comparative analysis. In addition, two first-in-dog clinical trials were performed in dogs with melanoma and osteosarcoma using autologous and allogeneic NK cells, respectively, to establish safety and proof-of-concept of this manufacturing approach. RESULTS: Calculated cell counts, viability, killing, and cytokine secretion were equivalent or higher in expanded NK cells from canine PBMCs versus CD5-depleted cells, and immune phenotyping confirmed a CD3-NKp46+ product from PBMC-expanded cells at day 14. Transcriptomic analysis of expanded cell populations confirmed upregulation of NK activation genes and related pathways, and human NK cells using well-characterized NK markers closely mirrored canine gene expression patterns. Autologous and allogeneic PBMC-derived NK cells were successfully expanded for use in first-in-dog clinical trials, resulting in no serious adverse events and preliminary efficacy data. RNA sequencing of PBMCs from dogs receiving allogeneic NK transfer showed patient-unique gene signatures with NK gene expression trends in response to treatment. CONCLUSIONS: Overall, the use of unmanipulated PBMCs appears safe and potentially effective for canine NK immunotherapy with equivalent to superior results to CD5 depletion in NK expansion, activation, and cytotoxicity. Our preclinical and clinical data support further evaluation of this technique as a novel platform for optimizing NK immunotherapy in dogs.

A retrospective Italian Society of Veterinary Oncology (SIONCOV) study of 56 cats with appendicular osteosarcoma.

Osteosarcoma is the most common malignant primary bone cancer, but it is infrequently reported in cats. Feline appendicular osteosarcoma typically exhibits good prognosis when treated with surgery alone. A retrospective multi-institutional study was conducted to identify possible prognostic factors. Cats diagnosed with appendicular osteosarcoma were included if initial staging and follow-up information were available. Data including signalment, tumour characteristics, treatment modalities, and survival outcomes were collected and analysed. Fifty-six cats were included; the femur was the most frequently affected bone. Eight cats had distant metastasis at admission and an additional 9 developed metastatic disease during follow-up, resulting in an overall metastatic rate of 30%. Forty-nine (87.5%) cats underwent surgery, and 4 also received adjuvant chemotherapy. Among operated cats, median time to local progression (TTLP), time to distant progression and tumour-specific survival (TSS) were not reached. One- and 2-year survival rates were 66% and 55%, respectively. Seven (12.5%) cats received no treatment; 1- and 2-year survival rates were 25% and 0%, respectively. Operated cats had significantly longer TTLP (P < .001) and TSS (P = .001) compared with non-operated cats. Among operated cats, young age negatively impacted local tumour progression, while the presence of distant metastasis at diagnosis was associated with a higher risk of tumour-related death. This study reaffirms the good prognosis for cats with appendicular osteosarcoma undergoing surgery, but sheds light on some additional factors to consider. Accurate initial staging is recommended, as the metastatic rate may exceed many previous estimations. Surgery substantially extends survival time, whereas the role of chemotherapy remains uncertain.

Reconstruction surgery using polypropylene mesh after extensive resection of a costal osteosarcoma in a dog.

Osteosarcoma is the most common tumour that develops in the chest wall of dogs; an extensive excision is the treatment of choice. Various methods have been reported for reconstruction of chest wall defects following extensive excision. The objective of this report was to describe the complete resection of an extensive costal osteosarcoma with an extended resection of the ribs and part of the diaphragm in a dog. An 11-year-old neutered, male, miniature pinscher was presented with dyspnoea: An extensive mass was observed, stretching from the right chest wall to the abdominal wall. On computed tomography, the mass originated from the right 9th rib and exceeded the 6th rib on the cranial side and the 13th rib on the caudal side; it was compressing the lungs, diaphragm, liver, stomach and duodenum. When the patient's condition was medically stabilized, the tumour was removed from the right 9th rib. In consideration of the surgical margin, the 5th-13th ribs were excised, and the tumour was resected with the thoracoabdominal wall and part of the diaphragm. The missing thoracoabdominal wall and section of the diaphragm were reconstructed using two sheets of a polypropylene mesh. Postoperatively, flail chest was observed, although dyspnoea was not observed in the patient. Histopathological examination confirmed the diagnosis of osteosarcoma with a clean margin. Although 60.6 months have passed post-surgery, no metastasis has reoccurred. In this case, complete resection and reconstruction of the chest wall and diaphragm were achieved using a polypropylene mesh without fatal postoperative complications, despite extensive osteosarcoma resection.

Telangiectatic osteosarcoma in four dogs: Cytologic, histopathologic, cytochemical, and immunohistochemical findings.

Telangiectatic osteosarcoma is a rare variant of osteosarcoma histologically and clinically similar to hemangiosarcoma (HSA). This case series describes the imaging and cytologic features of four histologically confirmed telangiectatic osteosarcomas, including the use of cytochemical stains. Alkaline phosphatase (ALP) was applied to Wright-Giemsa-stained cytology slides, and Factor VIII immunohistochemistry was evaluated. Cytologic characteristics included atypical mesenchymal cells with evidence of acute and chronic hemorrhage. Telangiectatic osteosarcoma cases had positive ALP cytochemical staining, while control HSA cases were negative. Factor VIII immunohistochemistry was negative in telangiectatic osteosarcoma and positive in HSA. Cytologic diagnosis of telangiectatic osteosarcoma with positive ALP cytochemical staining can help differentiate this neoplasm from HSA.

Osteosarcoma of the wing in a sulfur-crested cockatoo.

A 26-year-old female sulfur-crested cockatoo (Cacatua galerita) was evaluated for vocalizing through the night and extending her right wing. Physical examination revealed a large, firm mass extending from the humerus to the distal aspect of the elbow. Computed tomography confirmed a large aggressive mass of the right distal humerus with a large soft tissue component, severe osteolysis, and adjacent periosteal proliferation. Fine-needle aspirates of the mass were most compatible with sarcoma, and osteosarcoma was prioritized. An unstained slide was treated with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase (NBT/BCIP) substrate for ALP detection and was strongly positive, confirming a diagnosis of osteosarcoma. A month later, the patient underwent wing amputation and arrested during recovery from anesthesia. Post-mortem examination and histopathology were consistent with osteosarcoma. This case report highlights a rare occurrence of osteosarcoma in a cockatoo as well as its cytologic and histologic features. Additionally, this report provides support for NBT/BCIP application in ALP-expressing tumors, a cytochemical stain that has been minimally investigated in avian species.

Outcomes of 35 dogs with craniomaxillofacial osteosarcoma treated with stereotactic body radiation therapy.

Canine craniomaxillofacial osteosarcoma (OSA) is most commonly treated surgically; however, in cases where surgery is not feasible or non-invasive treatment is desired, stereotactic body radiation therapy (SBRT) may be elected for local tumour control. In this study, we evaluated 35 dogs treated with SBRT. Nine dogs (26%) had calvarial, seven (20%) had mandibular and 19 (54%) had maxillary OSA. Median time to first event (TFE) was 171 days, and overall median survival time (MST) was 232 days. Site-specific MSTs were 144 days for mandible, 236 days for calvarium and 232 days for maxilla (p = .49). Pulmonary metastatic disease was observed in 12/35 (34%) patients and was detected pre-SBRT in six dogs (17%) and post-SBRT in the remaining six dogs (17%). Eighteen adverse events post-SBRT were documented. Per veterinary radiation therapy oncology group criteria, five were acute (14%) and three were late (9%) grade 3 events. Neurological signs in two dogs were suspected to be early-delayed effects. Cause of death was local progression for 22/35 (63%) patients, metastasis for 9/35 (26%) patients and unknown for four. On univariate analysis, administration of chemotherapy was associated with a longer TFE (p = .0163), whereas volume of gross tumour volume was associated with a shorter TFE (p = .023). Administration of chemotherapy and five fractions versus single fraction of SBRT was associated with increased survival time (p = .0021 and .049). Based on these findings, a treatment protocol incorporating chemotherapy and five fractions of SBRT could be considered for dogs with craniomaxillofacial OSA electing SBRT with careful consideration of normal tissues in the field.

Updates in Osteosarcoma.

Clinical care of osteosarcoma (OSA) in dogs has seen little change during the past 2 decades, relying on amputation and platinum-based chemotherapy for pain control and survival. Recent advancements offer hope for improved outcomes. Genomic research reveals shared genetic abnormalities between canine and human OSA. Multidimensional imaging provides valuable staging and prognostic information. Limb-sparing approaches including stereotactic body radiation therapy are routine. Ablative therapies such as microwave ablation and histotripsy show promise. Immunotherapy including cell therapy and immune checkpoint inhibition are available. Radiopharmaceuticals are tuned to target OSA cells directly. These innovations may enhance treatment and prognosis for dogs with OSA.

Single-Nuclei Multiome (ATAC + Gene Expression) Sequencing of a Primary Canine Osteosarcoma Elucidates Intra-Tumoral Heterogeneity and Characterizes the Tumor Microenvironment.

Osteosarcoma (OSA) is a highly aggressive bone tumor primarily affecting pediatric or adolescent humans and large-breed dogs. Canine OSA shares striking similarities with its human counterpart, making it an invaluable translational model for uncovering the disease's complexities and developing novel therapeutic strategies. Tumor heterogeneity, a hallmark of OSA, poses significant challenges to effective treatment due to the evolution of diverse cell populations that influence tumor growth, metastasis, and resistance to therapies. In this study, we apply single-nuclei multiome sequencing, encompassing ATAC (Assay for Transposase-Accessible Chromatin) and GEX (Gene Expression, or RNA) sequencing, to a treatment-naïve primary canine osteosarcoma. This comprehensive approach reveals the complexity of the tumor microenvironment by simultaneously capturing the transcriptomic and epigenomic profiles within the same nucleus. Furthermore, these results are analyzed in conjunction with bulk RNA sequencing and differential analysis of the same tumor and patient-matched normal bone. By delving into the intricacies of OSA at this unprecedented level of detail, we aim to unravel the underlying mechanisms driving intra-tumoral heterogeneity, opening new avenues for therapeutic interventions in both human and canine patients. This study pioneers an approach that is broadly applicable, while demonstrating significant heterogeneity in the context of a single individual's tumor.

Safety and clinical efficacy of an anti-PD-L1 antibody (c4G12) in dogs with advanced malignant tumours.

Immune checkpoint inhibitors (ICIs) have been developed for canine tumour treatment, and pilot clinical studies have demonstrated their antitumour efficacy in dogs with oral malignant melanoma (OMM). Although ICIs have been approved for various human malignancies, their clinical benefits in other tumour types remain to be elucidated in dogs. Here, we conducted a clinical study of c4G12, a canine chimeric anti-PD-L1 antibody, to assess its safety and efficacy in dogs with various advanced malignant tumours (n = 12) at the Veterinary Teaching Hospital of Hokkaido University from 2018 to 2023. Dogs with digit or foot pad malignant melanoma (n = 4), osteosarcoma (n = 2), hemangiosarcoma (n = 1), transitional cell carcinoma (n = 1), nasal adenocarcinoma (n = 1), B-cell lymphoma (n = 1), or undifferentiated sarcoma (n = 2) were treated with 2 or 5 mg/kg c4G12 every 2 weeks. Treatment-related adverse events of any grade were observed in eight dogs (66.7%), including elevated aspartate aminotransferase (grade 3) in one dog (8.3%) and thrombocytopenia (grade 4) in another dog (8.3%). Among dogs with target disease at baseline (n = 8), as defined by the response evaluation criteria for solid tumours in dogs (cRECIST), one dog with nasal adenocarcinoma and another with osteosarcoma experienced a partial response (PR), with an objective response rate of 25.0% (2 PR out of 8 dogs; 95% confidence interval: 3.2-65.1%). These results suggest that c4G12 is safe and tolerable and shows antitumor effects in dogs with malignant tumours other than OMM. Further clinical studies are warranted to identify the tumour types that are most likely to benefit from c4G12 treatment.

A case of a dog with mandibular extraskeletal osteosarcoma after long-term puncture extirpation of the salivary gland cyst in the mandible.

Background: Extraskeletal osteosarcoma, unlike skeletal osteosarcoma, is a rare malignant mesenchymal tumor with a soft tissue primary that has been reported to occur in a variety of soft tissues. Case Description: The case is a 14-year-old, unneutered male Miniature Pinscher, weighing 6.7 kg, who had been treated medically for more than 5 years with a management strategy of puncture extirpation of a salivary gland cyst in the mandible; 1 month earlier, the fluid retention could not be removed, and after a computerized tomography scan showed no lesion in the mandible adjacent to the mass lesion, surgical resection was performed. Conclusion: Previous reports of extraskeletal osteosarcoma from the salivary glands in dogs have been rare. However, treatment of a salivary gland cyst in the mandible by long-term puncture extirpation may be a potential predisposing factor for the development of extraskeletal osteosarcoma around the mandible.

Sensitivity of osteosarcoma cell lines to autophagy inhibition as determined by pharmacologic and genetic manipulation.

Pharmacologic inhibition of autophagy can be achieved using lysosomotropic agents such as hydroxychloroquine (HCQ) that interfere with fusion of the autophagosome to the lysosome thus preventing completion of the recycling process. The goal of the present study is to determine the sensitivity of eight canine (cOSA) and four human (hOSA) osteosarcoma tumour cell lines to antiproliferative and cytotoxic effects of lysosomal autophagy inhibitors, and to compare these results to the autophagy-dependence measured using a CRISPR/Cas9 live-cell imaging assay in OSA and other tumour cell lines. Antiproliferative and cytotoxic response to HCQ and Lys05 was determined using live cell imaging and YOYO-1 staining. CRISPR/Cas9 live cell imaging screen was done using species specific guide RNA's and transfection of reagents into cells. Response to autophagy core genes was compared to response to an essential (PCNA) and non-essential (FOXO3A) gene. cOSA and hOSA cell lines showed similar antiproliferative and cytotoxic responses to HCQ and Lys05 with median lethal dose (Dm ) values ranging from 4.6-15.8 µM and 2.1-5.1 µM for measures of anti-proliferative response, respectively. A relationship was observed between antiproliferative responses to HCQ and Lys05 and VPS34 CRISPR score with Dm values correlating with VPS34 response (r = 0.968 and 0.887) in a species independent manner. The results show that a subset of cOSA and hOSA cell lines are autophagy-dependent and sensitive to HCQ at pharmacologically-relevant exposures.

Osteosarcoma in a free-living yellow armadillo (Euphractus sexcinctus).

An adult male free-living yellow armadillo (Euphractus sexcinctus) was found by hunters and referred for clinical evaluation because of a tumour-like lesion on the carapace. The animal was lethargic and weak with severe dehydration, enophthalmos and cachexia, and was euthanized because of its very poor clinical condition. Necropsy revealed a whitish, exophytic, irregular and moist mass (7 × 6.5 × 1.5 cm) in the caudal third of the carapace. On cut section, the mass was hard, compact, irregular and whitish. Histopathology revealed a densely cellular, expansive, poorly delimited neoplasm composed of malignant mesenchymal cells arranged in islands and cords, interspersed by numerous areas of irregularly mineralized osteoid matrix. Neoplastic cells were intensely immunolabelled for vimentin. The diagnosis of a high-grade osteosarcoma of osteoblastic subtype was based on the clinical, gross, histopathological and immunohistochemical findings. This first report of an osteosarcoma in an armadillo expands the list of armadillo diseases and will assist the management of these animals by veterinarians.

Transcriptional profiling of canine osteosarcoma identifies prognostic gene expression signatures with translational value for humans.

Canine osteosarcoma is increasingly recognized as an informative model for human osteosarcoma. Here we show in one of the largest clinically annotated canine osteosarcoma transcriptional datasets that two previously reported, as well as de novo gene signatures devised through single sample Gene Set Enrichment Analysis (ssGSEA), have prognostic utility in both human and canine patients. Shared molecular pathway alterations are seen in immune cell signaling and activation including TH1 and TH2 signaling, interferon signaling, and inflammatory responses. Virtual cell sorting to estimate immune cell populations within canine and human tumors showed similar trends, predominantly for macrophages and CD8+ T cells. Immunohistochemical staining verified the increased presence of immune cells in tumors exhibiting immune gene enrichment. Collectively these findings further validate naturally occurring osteosarcoma of the pet dog as a translationally relevant patient model for humans and improve our understanding of the immunologic and genomic landscape of the disease in both species.