Canine Osteosarcoma Patients Research Articles

Development of OX40 agonists for canine cancer immunotherapy

SummaryRecent breakthroughs in cancer immunotherapy have provided unprecedented clinical benefits to human cancer patients. Cancer is also one of the most common causes of death in pet dogs. Thus, canine-specific immune therapies targeting similar signaling pathways can provide better treatment options for canine cancer patients. Here, we describe the development and characterization of two canine-specific anti-OX40 agonists to activate OX40 signaling. We show that canine OX40, like human OX40, is not expressed on resting T cells, and its expression is markedly increased on canine CD4 T cells and Tregs after stimulation with concanavalin A (Con-A). cOX40 is also expressed on tumor-infiltrating lymphocytes (TILs) in canine osteosarcoma patients. The canine-specific OX40 agonists strongly activates cPBMCs by increasing IFN-γ expression and do not require Fc receptor-mediated cross-linking for OX40 agonism. Together, these results suggest that cFcOX40L proteins are potent OX40 agonists and have the potential to enhance antitumor immunity in canine cancer patients.

Comparative oncology analysis using genomic profiling of canine cancers by tissue and liquid biopsy testing reveals actionable somatic alterations with orthologs in human cancers.

e15012 Background: Targeted therapies offer great potential for cancer treatment in humans; however, enrollment in clinical trials may be slow, particularly for rare cancer types. The high homology between human and canine cancer genomes provides an opportunity for comparative oncology analyses that can inform the development of therapeutics for the benefit of both species. Currently, there is no well-established canine cancer genomic database. This study presents preliminary findings from an ongoing initiative aimed at building a foundational database of canine cancer genomic variants and their human orthologs via tissue and liquid biopsy profiling of multiple cancer types. Methods: Matched blood and tissue samples were collected from an all-comers cohort of over 150 cancer-diagnosed, client-owned dogs undergoing surgical resection or tissue sampling by biopsy. Cancer types included those found in both humans and dogs, such as lymphoma, osteosarcoma, mammary gland tumors and melanoma. Blood samples were processed to obtain plasma and white blood cells, and all samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Somatic variants detected by liquid biopsy and tissue testing were mapped to human orthologs targeted by therapeutic agents approved by the FDA or listed in NCCN guidelines, and to orthologs with therapeutic or prognostic levels of evidence annotated in human oncology databases. Results: Somatic alterations were identified in over half of the tissue and/or liquid biopsy samples tested. Results from tissue and liquid biopsy showed high concordance, and liquid biopsy revealed additional alterations that may have been missed in single-site tissue biopsies due to tumor heterogeneity. Of the mutations identified in plasma and/or tissue but absent in matched white blood cells, 10% mapped to a human ortholog listed as a biomarker targeted by either an approved therapeutic agent or a therapeutic currently undergoing clinical trials; examples include NRAS, HRAS and AKT1 mutations. Over 10% of canine cancer patients also harbored orthologs of somatic TP53 mutations reported to be prognostic in multiple human cancers. In particular, TP53 mutations were found in > 20% of canine osteosarcoma patients, which aligns with prior findings in human osteosarcoma and highlights the potential of comparative oncology for cancer types that are rare in humans but common in dogs. Conclusions: The results confirm the homology between canine and human cancers and emphasize the potential to translate genomic knowledge for biomarker discovery and therapeutic development between species. The high concordance between tumor and liquid biopsy findings support the prospect of using a noninvasive blood test to expand the genomic characterization effort to much larger cohorts of cancer-diagnosed dogs.

Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle.

Osteosarcoma (OS) is a highly malignant bone tumour that has seen little improvement in treatment modalities in the past 30 years. Understanding what molecules contribute to OS biology could aid in the discovery of novel therapies. Extracellular vesicles (EVs) serve as a mode of cell-to-cell communication and have the potential to uncover novel protein signatures. In our research, we developed a novel pipeline to isolate, characterize, and profile EVs from normal bone and osteosarcoma tissue explants from canine OS patients. Proteomic analysis of vesicle preparations revealed a protein signature related to protein metabolism. One molecule of interest, PSMD14/Rpn11, was explored further given its prognostic potential in human and canine OS, and its targetability with the drug capzimin. In vitro experiments demonstrated that capzimin induces apoptosis and reduces clonogenic survival, proliferation, and migration in two metastatic canine OS cell lines. Capzimin also reduces the viability of metastatic human OS cells cultured under 3D conditions that mimic the growth of OS cells at secondary sites. This unique pipeline can improve our understanding of OS biology and identify new prognostic markers and molecular targets for both canine and human OS patients.

Evaluation of the Chemotherapy Drug Response Using Organotypic Cultures of Osteosarcoma Tumours from Mice Models and Canine Patients.

Simple SummaryOsteosarcoma is a bone cancer with 75% of cases occurring in people younger than 25 years old. 35–45% of patients demonstrate resistance to chemotherapeutics and critically, survival rates for osteosarcoma is only 10–30% for patients with metastases. Therefore, reliable and patient-specific drug testing modalities are needed. Organotypic slice culture consists of sections of tumours, which survive and preserve the tumours mechanical and cellular properties, thereby enabling personalised testing of drugs. This study aimed to characterise organotypic slice cultures of osteosarcoma bone tumours derived from mice and dogs and to use these models for testing of anti-tumoural drugs. This study reports the various cell constituents of the model and the maintenance of osteosarcoma organotypic cultures over several weeks. A significantly decreased sensitivity to chemotherapy in 3D organotypic culture relative to 2D monolayer was found, highlighting the need to test anti-cancer drugs in a more personalized and biomimetic manner.Improvements in the clinical outcome of osteosarcoma have plateaued in recent decades with poor translation between preclinical testing and clinical efficacy. Organotypic cultures retain key features of patient tumours, such as a myriad of cell types organized within an extracellular matrix, thereby presenting a more realistic and personalised screening of chemotherapeutic agents ex vivo. To test this concept for the first time in osteosarcoma, murine and canine osteosarcoma organotypic models were maintained for up to 21 days and in-depth analysis identified proportions of immune and stromal cells present at levels comparable to that reported in vivo in the literature. Cytotoxicity testing of a range of chemotherapeutic drugs (mafosfamide, cisplatin, methotrexate, etoposide, and doxorubicin) on murine organotypic culture ex vivo found limited response to treatment, with immune and stromal cells demonstrating enhanced survival over the global tumour cell population. Furthermore, significantly decreased sensitivity to a range of chemotherapeutics in 3D organotypic culture relative to 2D monolayer was observed, with subsequent investigation confirming reduced sensitivity in 3D than in 2D, even at equivalent levels of drug uptake. Finally, as proof of concept for the application of this model to personalised drug screening, chemotherapy testing with doxorubicin was performed on biopsies obtained from canine osteosarcoma patients. Together, this study highlights the importance of recapitulating the 3D tumour multicellular microenvironment to better predict drug response and provides evidence for the utility and possibilities of organotypic culture for enhanced preclinical selection and evaluation of chemotherapeutics targeting osteosarcoma.

Abstract 672: Prognostic significance of urokinase plasminogen activator system in a naturally occurring model of osteosarcoma

Abstract Introduction Human pediatric and canine osteosarcoma (OSA) are both highly metastatic primary bone cancers that share molecular and clinical similarities. Urokinase plasminogen activator (uPA) and its receptor uPAR promote invasion, metastasis and angiogenesis in multiple cancer types and have been shown to play critical roles in malignant characteristics of OSA in vitro, yet their clinical significance has not been investigated in OSA patients. Methods Serum samples of dogs with naturally-occurring appendicular OSA (n=26) and clinically healthy dogs (n=6) were analyzed using commercially available canine-specific ELISA and uPA activity kits. Dogs with OSA that presented without metastatic disease at diagnosis and received treatment with amputation plus adjuvant carboplatin chemotherapy were included. Survival data and patient characteristics were obtained from medical records. Differences in serum uPA/uPAR results between the groups was tested by t test or Wilcoxan rank sum test. Kaplan Meier survival curve and log rank test were used for survival analysis in the OSA cohort after subgrouping by X-Tyle software. Well-established prognostic factors and serum uPA/uPAR results were tested for prognostic significance by cox regression analysis in both univariate and multivariate analyses. Results Dogs with OSA had significantly higher serum uPA (p=0.008; 1905.4 vs 1439.5 pg/mL) but not uPAR (p=0.866) levels compared to healthy dogs. OSA-bearing dogs with high serum uPA had significantly shorter progression-free (p=0.003; 94 vs 266 days) and overall survival (p=0.01; 123 vs 338 days) than dogs with low uPA level. Similarly, dogs with high serum uPA activity had significantly shorter progression-free survival (p=0.043; 227 vs 266 days). Serum uPAR was not prognostic in this cohort. Multivariate analysis revealed increased serum uPA level (p=0.024) as a poor prognostic indicator in addition to other established prognostic factors in dogs such as humeral location (p=0.022) and increased monocyte count (p=0.015). Conclusions Circulating uPA quantity and activity are increased in canine OSA patients with more aggressive disease, indicating importance of the uPA system in OSA disease progression. While an OSA-associated increase in circulating uPAR was not evident, this receptor likely contributes to OSA malignancy subsequent to the increased uPA ligand production. Serum uPA may be an attractive prognostic biomarker in OSA and the up-regulated uPA/uPAR axis should be further investigated as a potential therapeutic target for this disease. Citation Format: Arata Matsuyama, Anthony J. Mutsaers. Prognostic significance of urokinase plasminogen activator system in a naturally occurring model of osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 672.

Evaluation of tumor immunity after administration of conditionally replicative adenoviral vector in canine osteosarcoma patients

Osteosarcoma is one among the most common neoplasms in dogs. Current treatments show limited efficacy and fail to prevent metastasis. Conditionally replicative adenoviruses (CRAd) replicate exclusively in targeted tumor cells and release new virus particles to infect additional cells. We proposed that OC-CAVE1 (CAV2 with the E1A promoter replaced with the osteocalcin promotor) may also enhance existing immunity against tumors by overcoming immune tolerance via exposure of new epitopes and cytokine signaling. Eleven client-owned dogs with spontaneously occurring osteosarcomas were enrolled in a pilot study. All dogs were injected with OC-CAVE1 following amputation of the affected limb or limb-sparing surgery. Dogs were monitored for viremia and viral shedding. There was minimal virus shedding in urine and feces by the 6th day and no virus was present in blood after 4 weeks. CAV-2 antibody-titers increased in all of the patients, post-CRAd injection. Immunological assays were performed to monitor 1) humoral response against tumors, 2) levels of circulatory CD11c + cells, 3) levels of regulatory T cells, and 4) cytotoxic activity of tumor specific T cells against autologous tumor cells between pre-CRAd administration and 4 weeks post-CRAd administration samples. Administration of the CRAd OC-CAVE1 resulted in alteration of some immune response parameters but did not appear to result in increased survival duration. However, 2 dogs in the study achieved survival times in excess of 1 year. Weak replication of OC-CAVE1 in metastatic cells and delay of chemotherapy following CRAd treatment may contribute to the lack of immune response and improvement in survival time of the clinical patients.

Canine osteosarcoma checkpoint expression correlates with metastasis and T-cell infiltrate

BackgroundImmune-targeted therapies are being successfully implemented into cancer clinical practice. In particular checkpoint inhibitors are employed to modulate the immune microenvironment of solid tumors. We sought to determine the expression of PD-L1, HVEM, and B7H3 in human and canine osteosarcoma, and correlate expression with clinical features and tumor infiltrating lymphocytes in naturally-occurring canine osteosarcoma. MethodsFlow cytometry was used to measure ligand surface expression of five human and three canine cell lines. Immunohistochemistry was utilized for expression of ligands and lymphocyte markers in thirty-seven treatment-naïve canine osteosarcoma patients. ResultsAll cell lines expressed all three ligands at variable levels in both species. Metastatic lesions were associated with higher expression of all three ligands in patient tumor samples. PD-L1 expression strongly correlated with B7H3 and HVEM expression, while HVEM and B7H3 were weakly correlated. Whereas peritumoral T-cell expression positively correlated with PD-L1 and HVEM tumor expression, the presence of T-cells intratumorally were rare. Furthermore, intratumor penetration by T-cells was greatest in metastatic lesions, despite log-fold increases in peritumoral T-cells.In summary, PD-L1, HVEM, and B7H3 are expressed in osteosarcoma, with metastatic disease lesions expressing higher levels. We show for the first time that these ligands expressed on osteosarcoma cells positively correlate with each other and the presence of peritumoral T cell infiltration. Furthermore, osteosarcoma appears to be an intratumoral immune desert with significant resistance to effector T cells. Multiple agents targeting checkpoints are in clinical practice, and may have immune modulating benefit in osteosarcoma.

Comparison of serum cytokine concentrations between healthy dogs and canine osteosarcoma patients at the time of diagnosis

Systemic immune responses in cancer patients are of tremendous importance, both to advance understanding of disease mechanisms and for development of new diagnostic testing. Minimal published information is available on the systemic cytokine response in canine osteosarcoma (OS) patients. The goal of this study was to investigate serum cytokine alterations present in OS patients at the time of diagnosis. Serum samples from 22 canine OS patients at the time of diagnosis and 18 healthy control dogs were evaluated via multiplex immunoassay for 14 analytes. Significant increases in serum interleukin-8 (IL-8) and interleukin-12p40 (IL-12p40) concentrations were found in OS patients when compared to healthy controls. The results correlate with several published studies on serum cytokine alterations in human OS patients. These data add to the growing body of knowledge on immunologic alterations in OS, including potential immunomodulatory therapy of canine patients, and support future studies on serum cytokine testing to investigate diagnostic and prognostic utility.

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.

Abstract 2179: Gene expression models for predicting doxorubicin response in canine osteosarcoma

Abstract Purpose: The use of gene expression models for predicting individual response to cancer therapy is a promising area of research for improving outcome. Osteosarcoma is the most common primary bone cancer in dogs, with an average 1 year survival after treatment. The co-expression extrapolation (COXEN) method has been established as a gene expression method for accurately predicting drug sensitivity in human cancer. Our purpose is to explore the ability of COXEN for the intra- or interspecies extrapolation of cancer cell line data for prediction of chemosensitivity in canine osteosarcoma patients. Methods: Gene expression and drug sensitivity data to doxorubicin (DOX) for the human NCI60 cell line panel were publicly available. Corresponding data was obtained and generated for the canine ACC29 cell line panel and canine osteosarcoma tumors via cell viability assays, hospital record search, and microarray gene expression analysis. Differentially expressed genes (DEGs) were identified in reference sets by Significance Analysis of Microarrays or t-test analysis between sensitive and resistant samples to DOX. Probe matching between species was done based on highest target sequence homology or by collapsing probes to gene level by selecting probes with maximum variance between samples. Hierarchical clustering was done using the CIMminer program. DEGs are filtered for genes sharing strong co-expression between reference and co-expression datasets, followed by prediction model building using the Misclassification Penalized Posterior (MiPP) algorithm. Results: The NCI60 and ACC29 panels are similarly sensitive to DOX with log GI50 molar ranges of -8.3 to -5.7 and -8.2 to -5.9, respectively. Samples from ACC29 and COS16 clustered according to DOX sensitivity 67 and 100% correctly when DEGs from NCI60 were used. NCI60-trained models using DEGs co-expressed with the ACC29 were 83% accurate in predicting sensitivity to DOX in ACC29 (p = 0.019, binomial). NCI60-trained models using DEGs co-expressed with COS49 were 67% accurate in predicting response to DOX in COS49 (p = 0.049, binomial, p = 0.045, Log Rank test). Models with NCI60 DEGs but co-expressed and built on COS16 were 73% accurate in COS33 (p = 0.026, binomial, p = 0.001, Log Rank test). ACC29-trained models using DEGs co-expressed with COS49 were 73% accurate in COS49 (p = 0.008, binomial). Models with ACC29 or ACCosteo DEGs but co-expressed and built on COS16 were each 70% accurate in COS33 (p = 0.047, binomial, p = 0.071 and 0.071, Log Rank test). Conclusions: Both human and canine COXEN models were able to accurately predict response of canine osteosarcoma patients to DOX. The best performing model involved identifying DEGs from human cell lines, co-expressing and building the model with dog tumors, suggesting that genomic strategies in canine oncology could benefit from the wealth of available human genomic data, and could provide validation for personalized medicine in human cancer. Citation Format: Jared S. Fowles, Kristen C. Brown, Ann M. Hess, Dawn L. Duval, Daniel L. Gustafson. Gene expression models for predicting doxorubicin response in canine osteosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2179. doi:10.1158/1538-7445.AM2015-2179

Abstract 398: Genome-wide comparison of matched canine osteosarcoma primary tumours and metastases by array comparative genomic hybridization.

Abstract Canine osteosarcoma is a common and highly aggressive cancer that shares many characteristics with human osteosarcoma and can serve as a model of this disease. We collected snap frozen normal muscle, primary appendicular skeleton tumour, and matched terminal lung metastases from 8 canine osteosarcoma patients. DNA was isolated from these samples and gene copy number aberrations were assessed using high-resolution oligonucleotide array comparative genomic hybridization (Agilent G3 Canine 180k array). Data analysis was performed using Agilent Genomic Workbench 5.0 analysis software and the Aberration Detection Method 2 and Circular Binary Segmentation algorithms were applied to identify aberrant chromosomal intervals with the log2 ratio threshold set to 0.5. As expected, many cases had copy number aberrations that contain genes commonly altered in canine and human osteosarcoma, including MYC, CDKN2A/CDKN2B, PTEN, p53, and RB. To assess the degree of genomic similarity of the 8 primary tumours to their corresponding metastasis, we used these same samples to generate 8 random pairings of unmatched primaries and metastases, as well as 8 random pairs of primary tumours and 8 random pairs of metastases. A set of 11,000 random 10 base pair segments spanning the entire genome was generated and a correlation analysis was conducted between pairs of samples using the log2 ratios for each segment. Matched samples showed significant correlation with the square of the correlation coefficient (R2) being 0.44 (p<0.05), whereas unmatched primaries and metastases, random pairs of primaries, and random pairs of metastases did not show any significant correlation (R2= 0.08, 0.08, and 0.06 respectively, p>0.05 for each). Thus, as a group, primary tumours are genomically more similar to their corresponding metastasis than to unmatched metastases, and are also more similar than primary tumours or metastases are to each other. However, the range of similarity between matched primaries and metastases was relatively wide, with half the cases showing very high similarity and half showing moderate similarity that was close to the upper range of unmatched samples. These results have implications for therapies that rely on analysis of primary tumours to derive strategies that target metastases. Citation Format: Jonathan H W Liu, Yang Washington Shao, Sam D. Molyneux, Rama Khokha, Geoffrey A. Wood. Genome-wide comparison of matched canine osteosarcoma primary tumours and metastases by array comparative genomic hybridization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 398. doi:10.1158/1538-7445.AM2013-398

Abstract 2890: NCI60-based gene expression models for predicting chemosensitivity in canine cancer.

Abstract Background: Personalized medicine employing treatment strategies based on molecular characteristics of patient tumors is a promising area of research. Strong experimental validation of such methods is essential before initiating clinical trials involving patients receiving personalized treatment versus standard of care. The recently developed co-expression extrapolation (COXEN) method has proven successful in predicting drug sensitivity in vitro and in vivo in human cancer. Biologic and genetic similarities make canine cancer models highly attractive for human translational research. Our purpose in this study is to explore the COXEN method's utility in a cross-species extrapolation of gene expression models (GEMs) to predict chemosensitivity in canine cancer cells from a human reference set. Materials and Methods: Microarray gene expression and drug sensitivity data for doxorubicin (DOX), vinblastine (VBL), carboplatin (CAR), paclitaxel (PTX), lomustine (CCNU), and cisplatin (CIS) was publicly available for the human NCI60 panel. Gene expression data from 15 canine osteosarcoma (COS) tumors from dogs treated with DOX, CARBO, or CIS was obtained. Microarray gene expression analysis was performed for 16 canine cancer cell lines (ACC16). Sensitivity of the ACC16 to these chemotherapeutics was calculated via Alamar Blue assays. Significance Analysis of Microarrays (SAM) with a false discovery rate of 0.1 was performed on the 12 most sensitive and resistant cell lines from the NCI60 to identify gene expression signatures predicting sensitivity for each drug. Corresponding expression data from the ACC16 was compared to the NCI60 signatures via correlation matrices to identify co-expressed genes for prediction model development using the Misclassification Penalized Posterior (MiPP) algorithm. Results: GI50 ranges for all 6 drugs in the NCI60 panel were comparable to the ranges calculated for the ACC16. COXEN analysis generated GEMs for each drug that on average were 79% accurate in predicting sensitivity in the ACC16. GEMs for VBL, PTX, and CCNU resulted in COXEN prediction scores that significantly correlated with actual GI50 data (R=0.70, 0.78, 0.70 and p=0.02, 0.01, 0.03, Spearman). COXEN GEMs based on co-expressed genes between the NCI60 and COS tumors were 73% accurate in predicting disease free interval (DFI), as opposed to GEMs based on the NCI60 and ACC16, which were 47% accurate in DFI prediction in COS patients. Conclusions: Initial results are encouraging despite the limited sample set for the use of COXEN in cross-species prediction models and for future use in veterinary clinical trials. Future and ongoing studies will expand the ACC16 panel and generate microarray data for 50 additional COS tumor samples. Incorporating dog models in these genomic strategies may provide powerful validation that is needed for the continuing advancement of personalized medicine in cancer. Citation Format: Jared S. Fowles, Ann M. Hess, Dawn L. Duval, Daniel L. Gustafson. NCI60-based gene expression models for predicting chemosensitivity in canine cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2890. doi:10.1158/1538-7445.AM2013-2890

Fluoroquinolone-Mediated Inhibition of Cell Growth, S-G2/M Cell Cycle Arrest, and Apoptosis in Canine Osteosarcoma Cell Lines

Despite significant advancements in osteosarcoma research, the overall survival of canine and human osteosarcoma patients has remained essentially static over the past 2 decades. Post-operative limb-spare infection has been associated with improved survival in both species, yet a mechanism for improved survival has not been clearly established. Given that the majority of canine osteosarcoma patients experiencing post-operative infections were treated with fluoroquinolone antibiotics, we hypothesized that fluoroquinolone antibiotics might directly inhibit the survival and proliferation of canine osteosarcoma cells. Ciprofloxacin or enrofloxacin were found to inhibit p21WAF1 expression resulting in decreased proliferation and increased S-G2/M accumulation. Furthermore, fluoroquinolone exposure induced apoptosis of canine osteosarcoma cells as demonstrated by cleavage of caspase-3 and PARP, and activation of caspase-3/7. These results support further studies examining the potential impact of quinolones on survival and proliferation of osteosarcoma.

Wnt/β-Catenin Expression Does Not Correlate with Serum Alkaline Phosphatase Concentration in Canine Osteosarcoma Patients

Osteosarcoma is an aggressive malignancy of the bone and an increase in serum alkaline phosphatase concentration has clinical prognostic value in both humans and canines. Increased serum alkaline phosphatase concentration at the time of diagnosis has been associated with poorer outcomes for osteosarcoma patients. The biology underlying this negative prognostic factor is poorly understood. Given that activation of the Wnt signaling pathway has been associated with alkaline phosphatase expression in osteoblasts, we hypothesized that the Wnt/β-catenin signaling pathway would be differentially activated in osteosarcoma tissue based on serum ALP status. Archived canine osteosarcoma samples and primary canine osteosarcoma cell lines were used to evaluate the status of Wnt/β-catenin signaling pathway activity through immunohistochemical staining, western immunoblot analyses, quantitative reverse-transcription polymerase chain reaction, and a Wnt-responsive promoter activity assay. We found no significant difference in β-catenin expression or activation between OSA populations differing in serum ALP concentration. Pathway activity was mildly increased in the primary OSA cell line generated from a patient with increased serum ALP compared to the normal serum ALP OSA cell line. Further investigation into the mechanisms underlying differences in serum ALP concentration is necessary to improve our understanding of the biological implications of this negative prognostic indicator.

Nuclear scanning with99mTc‐HDP for the initial evaluation of osseous metastasis in canine osteosarcoma

The purpose of this retrospective analysis was to evaluate the use of nuclear scintigraphy in determining the rate of secondary sites of osseous malignancy at initial presentation in dogs with osteosarcoma. Radiographs of suspicious secondary lesions were reviewed and placed into four separate categories: benign lesions; no lesion seen on radiographs; subtle radiographic changes suggestive of, but not conclusive for, metastasis; and metastatic lesions highly suspected on radiographs. Three hundred and ninety-nine dogs were evaluated by technetium nuclear scanning for suspected osteosarcoma. Three hundred and twenty-six of 399 dogs (82%) had only one apparent site on the nuclear scan, whereas 72 dogs (18%) had more than one suspicious site on the nuclear scans. Highly suspected secondary metastatic lesions were detected by nuclear scans in 7.8% of cases. Although interpretation of nuclear scans is subjective, this study showed a 7.8% chance of detecting unsuspected osseous metastasis with nuclear scans in canine osteosarcoma patients on initial presentation.

Update on the biology and management of canine osteosarcoma.

Osteosarcoma (OSA) is the most common primary bone tumor diagnosed in dogs. Our understanding of the risk factors and genetic changes in canine OSA patients is growing, but specific, innovative therapeutic strategies are slow in coming. Appendicular skeletal osteosarcoma, the most frequent form of this disease, is typically seen in large to giant breeds, with males being overrepresented in most reports. Axial skeletal OSA is less common than appendicular OSA, but the biologic behavior of the disease is equally aggressive in all skeletal sites except for the mandible. Though the current standard of care for dogs with osteosarcoma remains surgical resection of the affected site, followed by chemotherapy with either a platinum- (cisplatin or carboplatin) or doxorubicin-based protocol, novel therapies are being actively investigated.