ArticleLiterature Review

Canine Osteosarcoma: A Naturally Occurring Disease to Inform Pediatric Oncology

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Abstract

Osteosarcoma (OSA) is the most common form of malignant bone cancer in children and dogs, although the disease occurs in dogs approximately 10 times more frequently than in people. Multidrug chemotherapy and aggressive surgical techniques have improved survival; however, new therapies for OSA are critical, as little improvement in survival times has been achieved in either dogs or people over the past 15 years, even with significant efforts directed at the incorporation of novel therapeutic approaches. Both clinical and molecular evidence suggests that human and canine OSA share many key features, including tumor location, presence of microscopic metastatic disease at diagnosis, development of chemotherapy-resistant metastases, and altered expression/activation of several proteins (e.g. Met, ezrin, phosphatase and tensin homolog, signal transducer and activator of transcription 3), and p53 mutations, among others. Additionally, canine and pediatric OSA exhibit overlapping transcriptional profiles and shared DNA copy number aberrations, supporting the notion that these diseases are similar at the molecular level. This review will discuss the similarities between pediatric and canine OSA with regard to histology, biologic behavior, and molecular genetic alterations that indicate canine OSA is a relevant, spontaneous, large animal model of the pediatric disease and outline how the study of naturally occurring OSA in dogs will offer additional insights into the biology and future treatment of this disease in both children and dogs.

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... Osteosarcoma (a form of bone cancer) occurs in both humans and dogs, its incidence in the latter being much higher (∼10 times) [73,80,81]. Osteosarcoma spreads rapidly, usually to the lungs, with treatment options typically limited to chemotherapy and amputation of the affected limb [81], although in dogs limb salvage with endoprostheses surgery is also available [82]. ...
... Osteosarcoma (a form of bone cancer) occurs in both humans and dogs, its incidence in the latter being much higher (∼10 times) [73,80,81]. Osteosarcoma spreads rapidly, usually to the lungs, with treatment options typically limited to chemotherapy and amputation of the affected limb [81], although in dogs limb salvage with endoprostheses surgery is also available [82]. Clinical and molecular evidence suggests that some features and risk factors are shared between humans and dogs [81] whilst noted differences can serve to improve cross-species studies, thus maximising potential for safe and effective strategies which could prevent its occurrence in both species [83]. ...
... Osteosarcoma spreads rapidly, usually to the lungs, with treatment options typically limited to chemotherapy and amputation of the affected limb [81], although in dogs limb salvage with endoprostheses surgery is also available [82]. Clinical and molecular evidence suggests that some features and risk factors are shared between humans and dogs [81] whilst noted differences can serve to improve cross-species studies, thus maximising potential for safe and effective strategies which could prevent its occurrence in both species [83]. ...
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The COVID-19 pandemic has resulted in the global recognition for greater inter-disciplinary and multi-disciplinary working, and the need for systematic approaches which recognise the interconnectedness and interactions between human, animal and environmental health. The notion of such a One Team/One science approach is perhaps best exemplified by the One Health concept, a systematic approach which is rapidly entering into the mainstream. However, the concept of One Health, as we presently know it, originated from One Medicine, a notion which is much older and which emerged to promote collaboration between the human and veterinary medicine professions and the allied health/scientific disciplines. Whilst One Medicine is perhaps better known by the veterinary community, some misconceptions of what One Medicine is have arisen. Therefore, this review introduces this emerging concept and how it can help to address overlapping (communicable and non-communicable disease) health challenges faced by both human and veterinary medicine.
... Osteosarcoma is an aggressive bone neoplasm occurring in dogs, which generally presents as lameness or pain associated with a bony or soft tissue mass or swelling [1]. Pathological fracture is reported to occur in 38% of osteosarcoma cases [2,3]. ...
... Treatment for osteosarcoma can include amputation of the affected limb or resection of axial lesions, and adjuvant chemotherapy may be recommended [2,4,5]. However, osteosarcoma often undergoes early haematogenous spread and, whilst just 10% of canine osteosarcoma cases present with gross metastases, 90% have been shown to possess microscopic metastatic disease at the time of diagnosis [1,2,4,6]. Therefore whilst amputation is appropriately carried out as palliative surgery to relieve pain, it is unable to prevent the metastatic spread which has already occurred in most osteosarcoma cases, and amputation therefore has little effect on survival. The lungs are the most common site of metastatic spread in canine osteosarcoma, and the median 1 year survival for dogs treated with amputation and chemotherapy is 45-50% [4,[6][7][8]. ...
... The appendicular skeleton (limbs and pelvis) represents the most common site of disease in large-breed dogs (95% of cases), whereas although only 5% of total osteosarcoma is reported to occur in dogs less than 15 kg, more than 65% of small-breed osteosarcomas are located in the axial skeleton (head, cervical and spinal vertebrae, sternum and ribs) [2,4,6]. A study of 85 appendicular osteosarcoma cases reported the most common lesion locations as the proximal humerus (26% of lesions), the distal radius (24%) and the distal tibia (15%), and multiple other analyses support this lesion distribution [1,2,4,9]. It is rare for osteosarcoma to be located as a mid-shaft lesion on any bone or near the elbow [2]. ...
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Background Osteosarcoma is an aggressive and painful bone neoplasm in dogs. Previous studies have reported epidemiological associations suggesting that large body mass, long bone length and the genetics of certain breeds including the Rottweiler are associated with elevated osteosarcoma risk. However, these studies were often limited by selection bias and confounding factors, and have rarely offered insights into breed-associated protection for osteosarcoma. The current study includes 1756 appendicular and axial osteosarcoma cases presenting to VPG Histology (Bristol, UK) compared against a control population of 905,211 dogs without osteosarcoma from primary care electronic patient records in the VetCompass™ dataset. Methods and study design Retrospective, case-control study. Multivariable logistic regression analysis explored associations between demographic risk factors (including breed, chondrodystrophy, age, sex/neuter status, skull-shape, and body mass) and osteosarcoma of all anatomical sites. Results We identified several breeds with increased and reduced odds of osteosarcoma. At highest risk were the Rottweiler and Great Dane, with > 10 times the odds of osteosarcoma compared with crossbreds, and the Rhodesian Ridgeback, which has not featured in previous lists of at-risk breeds for osteosarcoma, and had an odds ratio of 11.31 (95% confidence interval 7.37–17.35). Breeds at lowest risk of osteosarcoma (protected breeds) included the Bichon Frise, the French Bulldog and the Cavalier King Charles Spaniel, all with odd ratios of less than 0.30 compared with crossbreds. Body mass was strongly associated with osteosarcoma risk; dogs over 40 kg exhibited osteosarcoma odds of 45.44 (95% confidence interval 33.74–61.20) compared with dogs less than 10 kg. Chondrodystrophic breeds had an osteosarcoma odds ratio of 0.13 (95% confidence interval 0.11–0.16) compared with non-chondrodystrophic breeds. Conclusions This study provides evidence of strong breed-associated osteosarcoma risk and protection, suggesting a genetic basis for osteosarcoma pathogenesis. It highlights that breeds selected for long legs/large body mass are generally overrepresented amongst at-risk breeds, whilst those selected for short leg length/small body mass are generally protected. These findings could inform genetic studies to identify osteosarcoma risk alleles in canines and humans; as well as increasing awareness amongst veterinarians and owners, resulting in improved breeding practices and clinical management of osteosarcoma in dogs.
... Osteosarcoma (OS) is an aggressive malignancy of the bone that has a high metastatic rate [1]. Although the incidence of OS is rare in humans, accounting for fewer than 1000 cases per year in the USA, the incidence is notably higher in dogs and, in 2007, was estimated to be more than 8000 dogs each year [2]. ...
... Given the similarities in clinical presentation and molecular alterations between canine and human OS, canines have been highly regarded as a relevant, naturally occurring translational model for the human disease [1,46]. To supplement the clinical association we saw in published human datasets ( Figure 3A), and determine capzimin's effect on human OS cells, we treated the highly metastatic MG63.3 cell line with capzimin under standard 2D vs. 3D culture conditions. ...
... Upon owner consent and immediately following limb amputation, OS samples were obtained using sterile biopsy tools, while normal bone samples were obtained using a sterilized 1 2 , 1 4 , and 3/8-inch Milwaukee Diamond Plus hole saw drill bit from the opposite region of same bone (NB same ). A phalangeal bone sample was also obtained as a second normal bone sample (NB phal ) using a sterile scalpel blade. ...
Article
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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.
... However, depending on the proportion between females and males, mammary tumors have been observed around in about 36% of total cases (19), since mammary tumors account for more than 50% of the diagnosed tumors in females (20)(21)(22)(23). Other common types of cancer in dogs are located in the soft tissues (14), hematopoietic, and lymphoid tissues (18), digestive organs (19), and bones (24). ...
... Regarding genetic and signaling-pathway alterations, many types of canine cancer show similarities with their respective types of cancer in humans. Both human and canine osteosarcoma tumors carry mutations in tumor suppressor genes such as p53 (24,30,31) and RB1 (32) besides alterations in oncogenes expression including MYC and MET and constitutive expression of STAT3 (24,33). Overexpression of MYC, a consequence of copy number aberrations, can also be observed in both human and canine lymphomas (34,35). ...
... Regarding genetic and signaling-pathway alterations, many types of canine cancer show similarities with their respective types of cancer in humans. Both human and canine osteosarcoma tumors carry mutations in tumor suppressor genes such as p53 (24,30,31) and RB1 (32) besides alterations in oncogenes expression including MYC and MET and constitutive expression of STAT3 (24,33). Overexpression of MYC, a consequence of copy number aberrations, can also be observed in both human and canine lymphomas (34,35). ...
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A plethora of data has highlighted the role of epigenetics in the development of cancer. Initiation and progression of different cancer types are associated with a variety of changes of epigenetic mechanisms, including aberrant DNA methylation, histone modifications, and miRNA expression. At the same time, advances in the available epigenetic tools allow to investigate and reverse these epigenetic changes and form the basis for the development of anticancer drugs in human oncology. Although human and canine cancer shares several common features, only recently that studies emerged investigating the epigenetic landscape in canine cancer and applying epigenetic modulators to canine cancer. This review focuses on the existing studies involving epigenetic changes in different types of canine cancer and the use of small-molecule inhibitors in canine cancer cells.
... Canine and human OS share several key features such as presence of micrometastatic disease at diagnosis, p53 mutations, abnormal expression of several proteins (e.g., activator of transcription 3, tensin homolog, Met, phosphatase, signal transducer and ezrin), affected site and development of chemotherapy-resistance (28). Furthermore, OS in dogs and humans share similar DNA copy number aberrations and show overlapping transcriptional profiles, suggesting that these two diseases are similar at the molecular level. ...
... Furthermore, OS in dogs and humans share similar DNA copy number aberrations and show overlapping transcriptional profiles, suggesting that these two diseases are similar at the molecular level. In addition, the metastatic rate of OS without chemotherapy is 90% for dogs and 85-90% for humans and occur mostly in lung, bone and soft tissues, in both species (28). ...
... The high metastasis rate of OS results from the primary bone tumor spread via hematogenous path to other secondary locations (28). The most common cause of death in OS patients is the development of pulmonary metastasis (28). ...
Article
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Osteosarcoma (OS) is the most common primary bone tumor and originates from bone forming mesenchymal cells and primarily affects children and adolescents. The 5-year survival rate for OS is 60 to 65%, with little improvement in prognosis during the last four decades. Studies have demonstrated the evolving roles of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation, bone remodeling, regulation of calcium transport from blood to milk, regulation of maternal calcium transport to the fetus and reabsorption of calcium in kidneys. These two molecules also play critical roles in the development, progression and metastasis of several tumors such as breast cancer, lung carcinoma, chondrosarcoma, squamous cell carcinoma, melanoma and OS. The protein expression of both PTHrP and PTHR1 have been demonstrated in OS, and their functions and proposed signaling pathways have been investigated yet their roles in OS have not been fully elucidated. This review aims to discuss the latest research with PTHrP and PTHR1 in OS tumorigenesis and possible mechanistic pathways. This review is dedicated to Professor Michael Day who died in May 2020 and was a very generous collaborator.
... Osteosarcoma (OSA) is the most common primary osseous malignancy of the canine patient; OSA is highly aggressive, both locally and systemically. 13,21 Canine OSA (cOSA) is commonly used as a model system for human (pediatric) OSA (hOSA) given strong parallels in biologic behavior and response to treatment. 9,70 OSA seeds the body with micrometastatic lesions (primarily to the lungs) that can avoid detection prior to removal of the primary tumor. ...
... 5,6,71 In hOSA, 68% of patients survive for 5 y, and that survival rate decreases dramatically to < 30% once metastasis is present. 13,14,54 Although the addition of chemotherapy to OSA treatment protocols > 30 y ago improved survival outcomes for cOSA and hOSA, these survival times have sadly remained stagnant. 6,8,53,54 OSA is a highly heterogeneous and genetically chaotic tumor type. ...
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Osteosarcoma (OSA) is a highly aggressive and metastatic neoplasm of both the canine and human patient and is the leading form of osseous neoplasia in both species worldwide. To gain deeper insight into the heterogeneous and genetically chaotic nature of OSA, we applied single-cell transcriptome (scRNA-seq) analysis to 4 canine OSA cell lines. This novel application of scRNA-seq technology to the canine genome required uploading the CanFam3.1 reference genome into an analysis pipeline (10X Genomics Cell Ranger); this methodology has not been reported previously in the canine species, to our knowledge. The scRNA-seq outputs were validated by comparing them to cDNA expression from reverse-transcription PCR (RT-PCR) and Sanger sequencing bulk analysis of 4 canine OSA cell lines (COS31, DOUG, POS, and HMPOS) for 11 genes implicated in the pathogenesis of canine OSA. The scRNA-seq outputs revealed the significant heterogeneity of gene transcription expression patterns within the cell lines investigated (COS31 and DOUG). The scRNA-seq data showed 10 distinct clusters of similarly shared transcriptomic expression patterns in COS31; 12 clusters were identified in DOUG. In addition, cRNA-seq analysis provided data for integration into the Qiagen Ingenuity Pathway Analysis software for canonical pathway analysis. Of the 81 distinct pathways identified within the clusters, 33 had been implicated in the pathogenesis of OSA, of which 18 had not been reported previously in canine OSA.
... 146 Because the natural history of the disease in both species is similar, canine osteosarcoma has been long proposed as a surrogate disease model to better understand its human counterpart. 147,148 In addition to an anatomical predilection for the long bones and shared patterns of metastasis, a few molecular traits are conserved between canine and human osteosarcoma. An initiating event, such as TP53 mutation, seems to be required, which allows replication of chaotic genomes and clonal outgrowth of tumors with highly disrupted and heterogeneous genomes. ...
... For example, the reported age-adjusted incidence of osteosarcoma in humans is between 0.2 and 4.5 per million, with most affected patients being children, adolescents, and young adults, 150 whereas in dogs, this disease is most commonly observed in adult and elderly individuals from large and giant breeds, with a lifetime risk of approximately one in five at the extreme. 146,148,[151][152][153] The strong association between size and risk of osteosarcoma in dogs illustrates a similar, albeit weaker, association between risk of osteosarcoma and size in humans, where it is more common in males than in females, and in children who are taller and have high birthweights. [154][155][156] Of additional interest, larger dogs age more rapidly than smaller dogs and have shorter lifespans. ...
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Cancer is among the most common causes of death for dogs (and cats) and humans in the developed world, even though it is uncommon in wildlife and other domestic animals. We provide a rationale for this observation based on recent advances in our understanding of the evolutionary basis of cancer. Over the course of evolutionary time, species have acquired and fine‐tuned adaptive cancer‐protective mechanisms that are intrinsically related to their energy demands, reproductive strategies, and expected lifespan. These cancer‐protective mechanisms are general across species and/or specific to each species and their niche, and they do not seem to be limited in diversity. The evolutionarily acquired cancer‐free longevity that defines a species’ life history can explain why the relative cancer risk, rate, and incidence are largely similar across most species in the animal kingdom despite differences in body size and life expectancy. The molecular, cellular, and metabolic events that promote malignant transformation and cancerous growth can overcome these adaptive, species‐specific protective mechanisms in a small proportion of individuals, while independently, some individuals in the population might achieve exceptional longevity. In dogs and humans, recent dramatic alterations in healthcare and social structures have allowed increasing numbers of individuals in both species to far exceed their species‐adapted longevities (by two to four times) without allowing the time necessary for compensatory natural selection. In other words, the cancer‐protective mechanisms that restrain risk at comparable levels to other species for their adapted lifespan are incapable of providing cancer protection over this recent, drastic, and widespread increase in longevity.
... In dogs and humans, increasing age is associated with higher cancer rates [6,43,44]. Recently, dogs have been considered as natural cancer models owing to their fast aging rate, selective breeding, similar environment exposure, and response to treatment comparable to humans [3,[45][46][47]. Based on genetic classification, epidemiology and disease progression, canine osteosarcoma has a high genetic similarity of that found in children [45,48]. ...
... Based on genetic classification, epidemiology and disease progression, canine osteosarcoma has a high genetic similarity of that found in children [45,48]. Osteosarcoma is one of the most common types of cancer in large breed dogs and ranked within the fourth most common malignancies [46]. Mutations on p53 tumor suppressor gene, loss of phosphatase and tensin homolog (PTEN), retinoblastoma (RB1) gene, and STAT3 [49] signaling alterations have been found in dogs and humans diagnosed with osteosarcoma [48,50,51]. ...
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Background: Several studies have highlighted both the extreme anticancer effects of Cryptotanshinone (CT), a Stat3 crippling component from Salvia miltiorrhiza, as well as other STAT3 inhibitors to fight cancer. Methods: Data presented in this experiment incorporates 2 years of in vitro studies applying a comprehensive live-cell drug-screening analysis of human and canine cancer cells exposed to CT at 20 μM concentration, as well as to other drug combinations. As previously observed in other studies, dogs are natural cancer models, given to their similarity in cancer genetics, epidemiology and disease progression compared to humans. Results: Results obtained from several types of human and canine cancer cells exposed to CT and varied drug combinations, verified CT efficacy at combating cancer by achieving an extremely high percentage of apoptosis within 24 hours of drug exposure. Conclusions: CT anticancer efficacy in various human and canine cancer cell lines denotes its ability to interact across different biological processes and cancer regulatory cell networks, driving inhibition of cancer cell survival.
... Osteosarcoma (OS) is the most common primary malignant bone tumor in both humans and dogs. In both species, appendicular OS is predominant, occurring most frequently in the metaphysis of long bones; less often, OS affects the axial skeleton (Longhi et al. 2006;Morello et al. 2011;Fenger et al. 2014). OS has an incidence of »1/100,000 in humans across all ages, with an increased incidence of up to 11/100,000 in the age group 15À19 y (Brodey 1979;Fenger et al. 2014;Luetke et al. 2014). ...
... In both species, appendicular OS is predominant, occurring most frequently in the metaphysis of long bones; less often, OS affects the axial skeleton (Longhi et al. 2006;Morello et al. 2011;Fenger et al. 2014). OS has an incidence of »1/100,000 in humans across all ages, with an increased incidence of up to 11/100,000 in the age group 15À19 y (Brodey 1979;Fenger et al. 2014;Luetke et al. 2014). The 5-year survival rate in humans with non-metastatic OS is 60%À70%, with an estimated 20%À30% long-term survival in patients with identifiable metastases at diagnosis (Friebele et al. 2015;Meazza and Scanagatta 2016). ...
Article
Osteosarcoma (OS) is a primary bone tumor affecting both dogs and humans. Histotripsy is a non-thermal, non-invasive focused ultrasound method using controlled acoustic cavitation to mechanically disintegrate tissue. In this study, we investigated the feasibility of treating primary OS tumors with histotripsy using a 500-kHz transducer on excised canine OS samples harvested after surgery at the Veterinary Teaching Hospital at Virginia Tech. Samples were embedded in gelatin tissue phantoms and treated with the 500-kHz histotripsy system using one- or two-cycle pulses at a pulse repetition frequency of 250 Hz and a dosage of 4000 pulses/point. Separate experiments also assessed histotripsy effects on normal canine bone and nerve using the same pulsing parameters. After treatment, histopathological evaluation of the samples was completed. To determine the feasibility of treating OS through intact skin/soft tissue, additional histotripsy experiments assessed OS with overlying tissues. Generation of bubble clouds was achieved at the focus in all tumor samples at peak negative pressures of 26.2 ± 4.5 MPa. Histopathology revealed effective cell ablation in treated areas for OS tumors, with no evidence of cell death or tissue damage in normal tissues. Treatment through tissue/skin resulted in generation of well-confined bubble clouds and ablation zones inside OS tumors. Results illustrate the feasibility of treating OS tumors with histotripsy.
... Osteosarcoma (OS) is a primary bone cancer and a devastating disease for both human and canine patients. It is the most common primary bone tumor in children and adolescents, as well as, in the dog [53,54]. Figure 1A shows a CT image of an osteosarcoma in the forelimb of a dog. ...
... These similarities include matching biological behavior, nearly identical histological features, shared global gene expression signatures, and similar responses to standard treatment regimens [55][56][57][58][59]. For example, most OS lesions in dogs and humans occur in the appendicular skeleton around the metaphyseal region of long bones, and the disease tends to be associated with large and/or tall individuals [54,55]. The anatomic similarities between the canine and human skeletal structures allow the dog to be an accurate model for histotripsy systems development and treatment evaluation studies. ...
Article
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New therapeutic strategies are direly needed in the fight against cancer. Over the last decade, several tumor ablation strategies have emerged as stand-alone or combination therapies. Histotripsy is the first completely non-invasive, non-thermal, and non-ionizing tumor ablation method. Histotripsy can produce consistent and rapid ablations, even near critical structures. Additional benefits include real-time image-guidance, high precision, and the ability to treat tumors of any predetermined size and shape. Unfortunately, the lack of clinically and physiologically relevant pre-clinical cancer models is often a significant limitation with all focal tumor ablation strategies. The majority of studies testing histotripsy for cancer treatment have focused on small animal models, which have been critical in moving this field forward and will continue to be essential for providing mechanistic insight. While these small animal models have notable translational value, there are significant limitations in terms of scale and anatomical relevance. To address these limitations, a diverse range of large animal models and spontaneous tumor studies in veterinary patients have emerged to complement existing rodent models. These models and veterinary patients are excellent at providing realistic avenues for developing and testing histotripsy devices and techniques designed for future use in human patients. Here, we provide a review of animal models used in preclinical histotripsy studies and compare histotripsy ablation in these models using a series of original case reports across a broad spectrum of preclinical animal models and spontaneous tumors in veterinary patients.
... Osteosarcoma is the most common primary bone tumor reported in both human and companion dog populations [5,6]. Canine osteosarcoma is a suitable model for human osteosarcoma due to their similar clinical presentation, molecular features and therapeutic responses, offering a unique opportunity for preclinical modelling to allow development of innovative therapies for human patients [7][8][9][10][11]. In dogs, the prognosis remains poor. ...
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Purpose: 12b80 combines doxorubicin bound to a bone targeting hydroxybisphosphonate vector using a pH-sensitive linker, designed to specifically trigger doxorubicin release in an acidic bone tumor microenvironment. This phase I study aimed to determine the safety and toxicity profiles of 12b80 in dogs with naturally occurring osteosarcoma, with the objective to translate findings from dogs to humans. Experimental design: Ten client-owned dogs with osteosarcoma were enrolled in an accelerated dose-titration design followed by 3 + 3 design. Dogs received three cycles of 12b80 intravenous injection at 4 mg/kg (n = 1), 6 mg/kg (n = 2), 8 mg/kg (n = 3), and 10 mg/kg (n = 4). Endpoints included safety, tolerability, maximum tolerated dose (MTD), and dose-limiting toxicity (DLT). Results: The MTD of 12b80 was 8 mg/kg (i.e., equivalent dose of doxorubicin of 110 mg/m2, range: 93-126). Most adverse events included grade ≤ 2 gastrointestinal disorders and hypersensitivity reactions. No hematological or cardiac DLT were observed at any dose tested. Conclusions: In dogs, 12b80 is overall well tolerated and expends the MTD of doxorubicin up to four times the standard dose of 30 mg/m2. These results demonstrate the potential therapeutic benefit of 12b80 in canine and human osteosarcoma.
... Although the incidence of the disease is about ten times higher in the dog population than in humans, OSA is also the most common form of malignant bone cancer in children (13). Both clinical and molecular evidence has shown that OSA shares many common characteristics in humans and dogs such as anatomical location, presence of histopathologically diagnosed metastatic disease, development of chemotherapyresistant metastases and altered expression/activation (5). ...
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Introduction Clinoptilolite has antiviral, antibacterial, anti-inflammatory, antidiabetic, and anticancer properties due to its biological activities. In various cancer cell culture studies, it has been reported effective against tumour cells and gave positive results in treatment of various tumours in dogs. No study was found on the effects of the nanoparticulate form, nanoclinoptilolite, on cancer cells. The aim of this study was to determine its cytotoxic and apoptotic effects in canine osteosarcoma (OSA) cell culture. Material and Methods Doses at 50% inhibitory concentration were determined by measuring the dose- and duration-dependent cytotoxicity of nanoclinoptilolite on canine D-17 osteosarcoma cells by methylthiazol tetrazolium (MTT) test for 24 h, 48 h, and 72 h. Murine caspase-3 and -7 activity and expression levels of the BAX and BCL2 genes were measured using RT-PCR to investigate the apoptotic effect. Results Nanoclinoptilolite decreased cell viability and induced caspase-3- and -7-mediated apoptosis in treated canine OSA cells. Furthermore, its application to canine OSA cells downregulated the expression of BCL2 and upregulated the expression of proapoptotic BAX . Conclusion Clinoptilolite, which was previously demonstrated to have anticancer properties, decreased cell viability effectively and rapidly and increased the apoptotic cell ratio in a novel use in nanoparticle form, exhibiting this effect by increasing the BAX/BCL2 ratio.
... Studies have revealed that human and canine osteosarcoma is exquisitely similar in biology and pathophysiology (Legare et al., 2011;Mueller et al., 2007). Consequently, canines serve as an optimal large animal model to study and treat osteosarcoma (Fenger et al., 2014). ...
Article
Background Immune-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. Methods Flow 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. Results All 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.
... Well-characterized canine models of von Willebrand disease, 3 Glanzmann thrombasthenia, 4-6 storage pool diseases, 7 and immunemediated thrombocytopenia 8 have been identified. Dogs also share with humans various neoplastic diseases, including spontaneous solid tumors and leukemias, 9,10 where the role of platelets in pathogenesis is under active investigation. ...
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Background Domestic dogs represent a translational animal model to study naturally occurring human disease. Proteomics has emerged as a promising tool for characterizing human platelet pathophysiology; thus a detailed characterization of the core canine activated platelet secretome (CAPS) will enhance utilization of the canine model. The objectives of this study were development of a robust, high throughput, label‐free approach for proteomic identification and quantification of the canine platelet (i) thrombin releasate proteins, and (ii) the protein subgroup that constitutes CAPS. Methods Platelets were isolated from 10 healthy dogs and stimulated with 50 nmol/L of γ‐thrombin or saline. Proteins were in‐solution trypsin‐digested and analyzed by nano–liquid chromatography–tandem spectrometry. Core releasate proteins were defined as those present in 10 of 10 dogs, and CAPS defined as releasate proteins with a significantly higher abundance in stimulated versus saline controls (corrected P < .05). Results A total of 2865 proteins were identified; 1126 releasate proteins were present in all dogs, 650 were defined as CAPS. Among the differences from human platelets were a canine lack of platelet factor 4 and vascular endothelial growth factor C, and a 10‐ to 20‐fold lower concentration of proteins such as haptoglobin, alpha‐2 macroglobulin, von Willebrand factor, and amyloid‐beta A4. Twenty‐eight CAPS proteins, including cytokines, adhesion molecules, granule proteins, and calcium regulatory proteins have not previously been attributed to human platelets. Conclusions CAPS proteins represent a robust characterization of a large animal platelet secretome and a novel tool to model platelet physiology, pathophysiology, and to identify translational biomarkers of platelet‐mediated disease.
... Several oncogenes have been amplified in both humans and pet dogs, including MET, FOS, IGF1R, PVT1/MYC, RUNX2 and HER2 [115]. However, unique features of osteosarcoma in pet dogs, such as mutations in the epigenetic regulator SETD2, and deletions in DMD, the gene encoding dystrophin, may help explain the more aggressive behavior recognized in canine osteosarcoma [116][117][118]. These canine-specific molecular alterations may inform on the biology of aggressive disease or pinpoint a unique molecular subtype of aggressive human osteosarcoma. ...
Article
Preclinical studies in rodent models have been a pivotal role in human clinical research, but many of them fail in the translational process. Spontaneous tumors in pet dogs have the potential to bridge the gap between preclinical models and human clinical trials. Their natural occurrence in an immunocompetent system overcome the limitations of preclinical rodent models. Due to its reasonable cellular, molecular, and genetic homology to humans, the pet dog represents a valuable model to accelerate the translation of preclinical studies to clinical trials in humans, actually with benefits for both species. Moreover, their unique genetic features of breeding and breed-related mutations have contributed to assess and optimize therapeutics in individuals with different genetic backgrounds. This review aims to outline four main immunotherapy approaches – cancer vaccines, adaptive T-cell transfer, antibodies, and cytokines –, under research in veterinary medicine and how they can serve the clinical application crosstalk with humans.
... Osteosarcoma is the most common primary bone tumor in both humans and dogs, with the disease incidence being as much as 30-50 times higher in the latter (1). The similarities of the disease in humans and dogs are well-described and include commonalities in underlying molecular biology, including gene expression and genetic mutations, histopathology, clinical presentation, disease progression, and response to therapy (2). Spontaneous osteosarcoma in the dog has been used extensively as a preclinical large animal model to evaluate new therapies for osteosarcoma in humans, including limb-sparing procedures (3)(4)(5), chemotherapy delivery (6,7), targeted therapeutics (8), and immunotherapeutics, including therapeutic vaccines and others (9)(10)(11). ...
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Osteosarcoma is the most common primary bone tumor in both humans and dogs. It is a highly metastatic cancer and therapy has not improved significantly since the inclusion of adjuvant chemotherapy into disease treatment strategies. Osteosarcoma is an immunogenic tumor, and thus development of immunotherapies for its treatment, especially treatment of microscopic pulmonary metastases might improve outcomes. NK cells are lymphocytes of the innate immune system and can recognize a variety of stressed cells, including cancer cells, in the absence of major histocompatibility complex (MHC)-restricted receptor ligand interactions. NK cells have a role in controlling tumor progression and metastasis and are important mediators of different therapeutic interventions. The core hypothesis of adoptive natural killer (NK) cell therapy is there exists a natural defect in innate immunity (a combination of cancer-induced reduction in NK cell numbers and immunosuppressive mechanisms resulting in suppressed function) that can be restored by adoptive transfer of NK cells. Here, we review the rationale for adoptive NK cell immunotherapy, NK cell biology, TGFβ and the immunosuppressive microenvironment in osteosarcoma, manufacturing of ex vivo expanded NK cells for the dog and provide perspective on the present and future clinical applications of adoptive NK cell immunotherapy in spontaneous osteosarcoma and other cancers in the dog.
... Dog OSs are also gaining attention in the study of the metastatic process. Several studies of comparative oncology have highlighted the genetical, biological, and clinical similarities between metastatic human and canine disease [40,[80][81][82], recognizing the value of dog OS as a reliable model to study novel therapeutic agents against metastatic progression [23]. For example, recent positive results in the control of metastatic progression were obtained in a first-in-dog clinical trial combining radiation therapy and immunotherapy with adoptive transfer of autologous natural killer cells expanded ex vivo [83]. ...
Article
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Osteosarcoma (OS) is a rare malignant primary tumor of mesenchymal origin affecting bone. It is characterized by a complex genotype, mainly due to the high frequency of chromothripsis, which leads to multiple somatic copy number alterations and structural rearrangements. Any effort to design genome-driven therapies must therefore consider such high inter- and intra-tumor heterogeneity. Therefore, many laboratories and international networks are developing and sharing OS patient-derived xenografts (OS PDX) to broaden the availability of models that reproduce OS complex clinical heterogeneity. OS PDXs, and new cell lines derived from PDXs, faithfully preserve tumor heterogeneity, genetic, and epigenetic features and are thus valuable tools for predicting drug responses. Here, we review recent achievements concerning OS PDXs, summarizing the methods used to obtain ectopic and orthotopic xenografts and to fully characterize these models. The availability of OS PDXs across the many international PDX platforms and their possible use in PDX clinical trials are also described. We recommend the coupling of next-generation sequencing (NGS) data analysis with functional studies in OS PDXs, as well as the setup of OS PDX clinical trials and co-clinical trials, to enhance the predictive power of experimental evidence and to accelerate the clinical translation of effective genome-guided therapies for this aggressive disease.
... To further delineate the functional involvement of exosomes in the progression of osteosarcoma, we sought to use a more tractable in vivo model system for exosome biomarker discovery. Given the molecular and biological similarities between human and canine osteosarcomas (20,21), a spontaneous canine model was used for our studies. Our immediate next experiments, shown in the supplementary information, were thus devoted to characterizing canine osteosarcoma-derived exosomes and to confirm their conserved roles in the biology of the disease (6, 7). ...
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Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal mRNAs. The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts ( SKA2, NEU1, PAF1, PSMG2, and NOB1 ) was validated in dogs with spontaneous osteosarcoma by qRT-PCR, while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups (“healthy”, “osteosarcoma”, “other bone tumor”, or “non-neoplastic disease”). Pre-treatment samples from osteosarcoma cases were used as the training set and a validation set from post-treatment samples was used for testing, classifying as “osteosarcoma–detected” or “osteosarcoma–NOT detected”. Dogs in a validation set whose post-treatment samples were classified as “osteosarcoma–NOT detected” had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof-of-concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response. Abstract Figure
... The incidence is expected to be even higher in large/giant breed dogs compared to the general dog population. Dogs are arguably the best animal model to study human OSA because of the many similarities [4][5][6][7][8]. Both cancers are histologically similar, metastatically aggressive, and are treated with therapies that include surgery and platinumbased chemotherapy [5]. ...
Article
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Osteosarcoma is a rare disease in children but is one of the most common cancers in adult large breed dogs. The mutational landscape of both the primary and pulmonary metastatic tumor in two dogs with appendicular osteosarcoma (OSA) was comprehensively evaluated using an automated whole genome sequencing, exome and RNA-seq pipeline that was adapted for this study for use in dogs. Chromosomal lesions were the most common type of mutation. The mutational landscape varied substantially between dogs but the lesions within the same patient were similar. Copy number neutral loss of heterozygosity in mutant TP53 was the most significant driver mutation and involved a large region in the middle of chromosome 5. Canine and human OSA is characterized by loss of cell cycle checkpoint integrity and DNA damage response pathways. Mutational profiling of individual patients with canine OSA would be recommended prior to targeted therapy, given the heterogeneity seen in our study and previous studies.
... It is a realistic possibility that pet dogs (Canis lupus familiaris) will play an expanding role in such testing, given the parallels of some naturally-arising canine cancers, such as osteosarcoma, glioma, urothelial carcinoma and non-Hodgkin lymphoma (NHL), to their human counterparts (reviewed in Refs. [1][2][3][4][5][6][7][8]. Studies with CAR T cells and PD-1/PD-L1 blockade have been reported. ...
Article
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Canine spontaneous cancers may offer greater fidelity than rodent models in advancing clinical immunotherapies. Boxers in particular are distinguished as study subjects by their popularity, and high incidence of human‐relevant cancers. Further, the MHC class I allele DLA‐88*034:01, with a known motif, dominates the breed, facilitating discovery of shared CTL responses against mutation‐origin neoepitopes by standard prediction methods. We experimentally confirmed the allomorph's binding motif by developing an MHC surface stabilization assay. The assay validated four DLA‐88‐034:01‐presented peptides from canine distemper virus, ubiquitously administered in routine vaccines, for positive controls in future CTL studies. In turn, these viral peptides substantiated motif‐based prediction for DLA‐88*034:01. The study adds new tools for studying neoepitope‐specific CTL in Boxers to foster canine comparative oncology. This article is protected by copyright. All rights reserved.
... For osteosarcoma, companion dogs are very comparable to pediatric patients since it is a common disease of both canine and pediatric patients [125]. Dogs develop spontaneous osteosarcomas with an incidence of ten times that of humans, thus dogs are an ideal model for pediatric osteosarcoma, representing a tumor developing spontaneously [126]. In both species, metastatic and relapsed disease have poor survival with current chemotherapy. ...
Article
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Treatment of metastatic pediatric solid tumors remain a significant challenge, particularly in relapsed and refractory settings. Standard treatment has included surgical resection, radiation, chemotherapy, and, in the case of neuroblastoma, immunotherapy. Despite such intensive therapy, cancer recurrence is common, and most tumors become refractory to prior therapy, leaving patients with few conventional treatment options. Natural killer (NK) cells are non-major histocompatibility complex (MHC)-restricted lymphocytes that boast several complex killing mechanisms but at an added advantage of not causing graft-versus-host disease, making use of allogeneic NK cells a potential therapeutic option. On top of their killing capacity, NK cells also produce several cytokines and growth factors that act as key regulators of the adaptive immune system, positioning themselves as ideal effector cells for stimulating heavily pretreated immune systems. Despite this promise, clinical efficacy of adoptive NK cell therapy to date has been inconsistent, prompting a detailed understanding of the biological pathways within NK cells that can be leveraged to develop “next generation” NK cell therapies. Here, we review advances in current approaches to optimizing the NK cell antitumor response including combination with other immunotherapies, cytokines, checkpoint inhibition, and engineering NK cells with chimeric antigen receptors (CARs) for the treatment of pediatric solid tumors.
... A series of studies have shown similarities between canine and human osteosarcomas [38][39][40] regarding the morphology and molecular alterations. In particular, canine osteosarcomas can be models for pediatric osteosarcomas [41]. Therefore, new drugs that can inhibit canine tumors, including osteosarcomas, can be tested in humans, eventually children, and represent novel therapies for these aggressive diseases. ...
Article
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Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.
... The predominant bone cancer diagnosed both in human and canine patient is OSA sharing common attributes like tumor location, presence of micrometastatic disease at diagnosis, altered expression of several proteins along with p53 mutations (Fenger et al., 2014). Hence, understanding the disease in canine models and development of new therapeutic approach will eventually lead to numerous drug formulations in humans. ...
Article
Background: Canine appendicular osteosarcoma (OSA) is the most common bone malignancy in dogs. Methods: Spontaneous cases of canine OSA presented for three years were treated with different standard therapeutic protocols and compared with a novel plant formulations or nutraceuticals prepared from combination of turmeric, clove and olive oil. Different diagnostic modalities like survey and three view thoracic radiograph, abdominal ultrasonography (USG) with spectral Doppler USG, greyscale USG, strain elastography, fine needle aspiration cytology (FNAC), computed tomography and excisional biopsy were used to detect appendicular OSA and for staging of the primary bone tumor.Result: Canine bone tumor of appendicular OSA primarily affects large breed dogs with median age 10.32 years (range, 2.6-13 years) and median body weight 32.95 kg (range, 14-41 kg). Among four treatment groups, disease-free interval (DFI) and overall survival times were longer in groups of amputation followed by carboplatin as adjuvant chemotherapy (9 no.) than in groups with neoadjuvant chemotherapy with carboplatin followed by amputation (2 no.) and amputation along with nutraceuticals treatment (6 no.) and were shorter in the control group with amputation alone (6 no.). Herbal nutraceuticals can be included in the therapeutic regimen of canine osteosarcoma for increasing the overall survival time.
... However, OSA incidence rates in dogs are about 27 times higher than in humans [4]. The higher incidence rate of canine OSA makes dog a good model for human disease [5]. OSA can affect appendicular or axial skeleton; nevertheless, it is most frequently found in the long bones of the extremities both in humans (up 90% of cases) and dogs (estimated up of 80% of cases) while in other species occurs most commonly in axial skeleton or flat bone [3,4]. ...
Article
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Osteosarcoma (OSA) is a rare cancer both in human and dog although the incidence rate in dogs is 27 times higher than in human. Many studies employed D-17 as cell line for in vitro test to evaluate conventional anticancer therapies; however, little is known about D-17 cell line. The aim of our study was to evaluate the basal level of gene expression of pivotal molecules in the innate immune response and cell cycle regulation and to establish the ability of this cell line to react to Salmonella typhimurium (ST) infective stressor. IL15, IL10, iNOS, TLR5, CD14, PTEN and IL18 were expressed in an inconsistent manner among experiments. The other genes under study were expressed in all samples. ST showed ability to penetrate D-17 causing pro-inflammatory response. Our results outline the expression in D-17 of important genes involved in innate immune response. These results provide important data on D-17 basal gene expression profile useful for in vitro preliminary evaluation of new therapeutic approaches.
... It shares many clinical and molecular features with human OSA (5)(6)(7)(8). The current management of choice for canine OSA is surgery followed by chemotherapy; the one year survival rarely exceeds 45% even for patients receiving treatment (5,(9)(10)(11)(12)(13)(14). In contrast to human OSA, canine OSA is most common in middle aged dogs and a degree of heritability has been observed (1,15,16). ...
Article
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Osteosarcoma (OSA) is an aggressive bone malignancy. Unlike many other malignancies, OSA outcomes have not improved in recent decades. One challenge to the development of better diagnostic and therapeutic methods for OSA has been the lack of well characterized experimental model systems. Spontaneous OSA in dogs provides a good model for the disease seen in people and also remains an important veterinary clinical challenge. We recently used RNA sequencing and qRT-PCR to provide a detailed molecular characterization of OSA relative to non-malignant bone in dogs. We identified differential mRNA expression of the solute carrier family 2 member 1 (SLC2A1/GLUT1), matrix metallopeptidase 3 (MMP3) and nuclear factor erythroid 2–related factor 2 (NFE2L2/NRF2) genes in canine OSA tissue in comparison to paired non-tumor tissue. Our present work characterizes protein expression of GLUT1, MMP3 and NRF2 using immunohistochemistry. As these proteins affect key processes such as Wnt activation, heme biosynthesis, glucose transport, understanding their expression and the enriched pathways and gene ontologies enables us to further understand the potential molecular pathways and mechanisms involved in OSA. This study further supports spontaneous OSA in dogs as a model system to inform the development of new methods to diagnose and treat OSA in both dogs and people.
... Dogs develop many of the same malignancies as humans, including osteosarcoma, lymphoma, gliomas and melanoma (9). In fact, the natural history, genetic mutations, and response to therapy of canine cancers are remarkably similar to humans (8)(9)(10)(11). ...
Article
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Natural killer (NK) cells are key effectors of the innate immune system, but major differences between human and murine NK cells have impeded translation. Outbred dogs offer an important link for studies of NK biology and immunotherapy. We analyzed gene expression of putative NK populations from healthy dogs and dogs with naturally-occurring cancers examining differential gene expression across multiple conditions, including steady-state, in vitro activation with cytokines and co-culture, and in vivo activation with inhaled IL-15 in dogs receiving IL-15 immunotherapy. We also compared dog, mouse and human CD3-NKp46+ NK cells using a novel orthologous transcriptome. Distinct transcriptional profiles between NK populations exist between conditions and in vitro versus in vivo treatments. In cross-species analysis, canine NK cells were globally more similar to human NK cells than mice. These data define canine NK cell gene expression under multiple conditions and across species, filling an important gap in translational NK studies.
... Canine and human OS cells share many dysregulated intracellular signaling pathways, allowing a human OS cell line (MG63.3) to reasonably represent canine disease [12]. The recognized genomic complexity and heterogeneity of OS, and our dependence on highly characterized cell lines is likely a greater weakness to the current approach than the absence of a canine cell line. ...
Article
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Osteosarcoma (OS) is an aggressive mesenchymal cell tumor that carries a poor long-term prognosis. Despite definitive surgery for the primary tumor and adjuvant chemotherapy, pulmonary metastasis is common and is the primary cause of morbidity. To improve outcomes for patients, we have developed and optimized a phenotypic screen for drugs that may target OS disseminated tumor cells (DTCs) and inhibit their metastatic outbreak rather than merely screening for cytotoxic activity against proliferating cells, as is commonly conducted in conventional drug discovery approaches. We report on the validation of a previously described 3D reconstituted basement membrane extract (3D BME) model system for tumor dormancy and metastatic outgrowth adapted to clonal pairs of high and low metastatic OS cells. A post-hoc validation of the assay was possible by comparing the activity of a drug in our assay with early evidence of activity in human OS clinical trials (regorafenib and saracatinib). In this validation, we found concordance between our assay and human clinical trial experience We then explored an approved veterinary small molecule inhibitor of Janus kinase-1 (oclacitinib) as a potential drug candidate to take advantage of the high prevalence of OS in pet dogs and its translational value to humans. Despite the biological rationale, we found no evidence to support the use of oclacitinib as an antimetastatic agent in OS. The findings support our 3D BME assay as a highly efficient method to examine drugs for activity in targeting OS DTCs
... Osteosarcoma incidence is~10 times more prevalent in dogs than in humans, thereby providing a significant patient cohort to test chemotherapeutic treatments [34,54]. The shorter canine lifespan and reduced survival times achieved in canine patients in response to combined surgery and cytotoxic chemotherapy allows for clinical trials in dogs to be conducted over a shorter time frame, facilitating more rigorous assessment of treatments before translation into human trials [55]. As proof of principle for ex vivo chemotherapeutic screening on patient-derived tumours, we tested doxorubicin efficacy on canine osteosarcoma tumours in organotypic culture for the first time. ...
Article
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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.
... For example, the BRAF gene's somatic mutation occurs in nearly 60% of melanoma from humans, but only in approximately 6% of dogs [125]. Also, while osteosarcoma most typically affects the appendicular skeleton and metastasizes to lungs in humans and dogs, peak onset occurs at a young age in humans, but more often at an advanced age in dogs [126]. ...
Chapter
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Over the history of humankind, knowledge acquisition regarding the human body, health, and the development of new biomedical techniques have run through some animal model at some level. The mouse model has been primarily used as the role model for a long time; however, it is severely hampered regarding its feasibility for translational outcomes, in particular, to preclinical and clinical studies. Herein we aim to discuss how induced pluripotent stem cells generated from non-human primates, pigs and dogs, all well-known as adequate large biomedical models, associated or not with gene editing tools, can be used as models on in vivo or in vitro translational research, specifically on regenerative medicine, drug screening, and stem cell therapy.
... Osteosarcoma is a primary malignant bone tumor that can occur in any age group but mainly affects children, adolescents, and young adults and can also occur in animals [49]. Cinegaglia et al. [23] demonstrated that the hydroethanolic geopropolis extract produced by Melipona fasciculata exerts an in vitro cytotoxic effect against canine osteosarcoma cells in a dose-and time-dependent manner (24, 48, and 72 h). ...
Article
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Cancer is one of the major maladies affecting humankind and remains one of the leading causes of death worldwide. The investigation of the biological activities of stingless bee products, especially propolis and geopropolis, has revealed promising therapeutic properties, especially in the research on new antineoplastic agents. This literature review of preclinical trials, involving biological assays of antitumor activity and identification of the chemical composition of propolis and geopropolis of stingless bee species, describes the cytotoxicity in tumor lineages (breast, lung, ovarian, liver, mouth, pharynx, larynx, colon, stomach, colorectal, cervix, kidney, prostate, melanoma, human glioblastoma, canine osteosarcoma, erythroleukemia, human chronic myelocytic leukemia, and human promyelocytic leukemia) of propolis and geopropolis of 33 species of stingless bees. The chemical composition of propolis and geopropolis was identified, indicating that these belong to the chemical classes of phenolic acids, flavonoids, coumarins, benzophenones, anthraquinones, alkaloids, terpenes, steroids, saponins, fatty acids, and carbohydrates and are possibly responsible for the cytotoxicity in tumor cells. Apoptosis was one of the main mechanisms of cytotoxicity of extracts and substances isolated from stingless bee products. Although the results found are encouraging, other preclinical studies and clinical trials are essential for the discovery of new anticancer agents.
... Osteosarcoma is a primary malignant bone tumor that can occur in any age group but mainly affects children, adolescents, and young adults and can also occur in animals [49]. Cinegaglia et al. [23] demonstrated that the hydroethanolic geopropolis extract produced by Melipona fasciculata exerts an in vitro cytotoxic effect against canine osteosarcoma cells in a dose-and time-dependent manner (24, 48, and 72 h). ...
... In the past few decades, there has been limited advancement of OSA therapies, and outcomes for patients with metastatic disease have remained stagnant (4,5). Canine OSA (cOSA) occurs spontaneously and shares notable genomic profiles, clinical presentations, and progression patterns with human OSA (hOSA) (1,(6)(7)(8). The intact immune system of dogs with naturally occurring cancer along with the relatively high incidence of cOSA and extensive similarities between cOSA and hOSA make companion dogs an ideal platform for translational oncology, especially in the investigation of novel immunotherapies (9,10). ...
Article
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Metastatic osteosarcoma has a bleak prognosis in both humans and dogs, and there have been minimal therapeutic advances in recent decades to improve outcomes. Naturally occurring osteosarcoma in dogs is shown to be a highly suitable model for human osteosarcoma, and limited data suggest the similarities between species extend into immune responses to cancer. Studies show that immune infiltrates in canine osteosarcoma resemble those of human osteosarcoma, and the analysis of tumor immune constituents as predictors of therapeutic response is a promising direction for future research. Additionally, clinical studies in dogs have piloted the use of NK transfer to treat osteosarcoma and can serve as valuable precursors to clinical trials in humans. Cytotoxic lymphocytes in dogs and humans with osteosarcoma have increased activation and exhaustion markers within tumors compared with blood. Accordingly, NK and T cells have complex interactions among cancer cells and other immune cells, which can lead to changes in pathways that work both for and against the tumor. Studies focused on NK and T cell interactions within the tumor microenvironment can open the door to targeted therapies, such as checkpoint inhibitors. Specifically, PD-1/PD-L1 checkpoint expression is conserved across tumors in both species, but further characterization of PD-L1 in canine osteosarcoma is needed to assess its prognostic significance compared with humans. Ultimately, a comparative understanding of T and NK cells in the osteosarcoma tumor microenvironment in both dogs and humans can be a platform for translational studies that improve outcomes in both dogs and humans with this frequently aggressive disease.
... The sporadic canine tumor model overcomes many of the rodent model deficiencies described here, and the pet dog with spontaneously occurring glioma may offer an ideal immune-competent model for cancer in humans. The clinical, anatomic, and histological similarities between canine and human cancer patients have been previously described [14][15][16] . Dogs respond to and similarly metabolize drugs to humans [17,18] . ...
Article
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Aim: To molecularly characterize the tumor microenvironment and evaluate immunologic parameters in canine glioma patients before and after treatment with oncolytic human IL-12-expressing herpes simplex virus (M032) and in treatment naïve canine gliomas. Methods: We assessed pet dogs with sporadically occurring gliomas enrolled in Stage 1 of a veterinary clinical trial that was designed to establish the safety of intratumoral oncoviral therapy with M032, a genetically modified oncolytic herpes simplex virus. Specimens from dogs in the trial and dogs not enrolled in the trial were evaluated with immunohistochemistry, NanoString, Luminex cytokine profiling, and multi-parameter flow cytometry. Results: Treatment-naive canine glioma microenvironment had enrichment of Iba1 positive macrophages and minimal numbers of T and B cells, consistent with previous studies identifying these tumors as immunologically "cold". NanoString mRNA profiling revealed enrichment for tumor intrinsic pathways consistent with suppression of tumor-specific immunity and support of tumor progression. Oncolytic viral treatment induced an intratumoral mRNA transcription signature of tumor-specific immune responses in 83% (5/6) of canine glioma patients. Changes included mRNA signatures corresponding with interferon signaling, lymphoid and myeloid cell activation, recruitment, and T and B cell immunity. Multiplexed protein analysis identified a subset of oligodendroglioma subjects with increased concentrations of IL-2, IL-7, IL-6, IL-10, IL-15, TNFα, GM-CSF between 14 and 28 days after treatment, with evidence of CD4+ T cell activation and modulation of IL-4 and IFNγ production in CD4+ and CD8+ T cells isolated from peripheral blood. Conclusion: These findings indicate that M032 modulates the tumor-immune microenvironment in the canine glioma model.
... Although rare in humans, OS is seen frequently in dogs, where they invariably demonstrate a rapid progression to metastatic disease and death [34,35]. In this study, we investigated differences in ALDH expression and serum Cu concentrations in human and canine sarcoma clinical samples from patients in our practices with known metastatic histories. ...
Article
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Aldehyde dehydrogenase 1A1 (ALDH) is a cancer stem cell marker highly expressed in metastatic cells. Disulfiram (Dis) is an FDA-approved antialcoholism drug that inhibits ALDH and has been studied as a candidate for drug repurposing in multiple neoplasia. Dis cytotoxicity in cancer cells has been shown to be copper-dependent, in part due to Dis’s ability to function as a bivalent metal ion chelator of copper (Cu). The objectives of this research were to test ALDH expression levels and Cu concentrations in sarcoma patient tumors and human osteosarcoma (OS) cell lines with differing metastatic phenotypes. We also sought to evaluate Dis + Cu combination therapy in human OS cells. Intracellular Cu was inversely proportional to the metastatic phenotype in human OS cell lines (SaOS2 > LM2 > LM7). Nonmetastatic human sarcoma tumors demonstrated increased Cu concentrations compared with metastatic tumors. qPCR demonstrated that ALDH expression was significantly increased in highly metastatic LM2 and LM7 human OS cell lines compared with low metastatic SaOS2. Tumor cells from sarcoma patients with metastatic disease displayed significantly increased ALDH expression compared with tumor cells from patients without metastatic disease. Serum Cu concentration in canine OS versus normal canine patients demonstrated similar trends. Dis demonstrated selective cytotoxicity compared with human multipotential stromal cells (MSCs): Dis-treated OS cells demonstrated increased apoptosis, whereas MSCs did not. CuCl2 combined with Dis and low-dose doxorubicin resulted in a superior cytotoxic effect in both SaOS2 and LM7 cell lines. In summary, ALDH gene expression and Cu levels are altered between low and highly metastatic human OS cells, canine samples, and patient tumors. Our findings support the feasibility of a repurposed drug strategy for Dis and Cu in combination with low-dose anthracycline to specifically target metastatic OS cells.
... A series of studies have shown similarities between canine and human osteosarcomas [38][39][40] regarding the morphology and molecular alterations. In particular, canine osteosarcomas can be models for pediatric osteosarcomas [41]. Therefore, new drugs that can inhibit canine tumors, including osteosarcomas, can be tested in humans, eventually children, and represent novel therapies for these aggressive diseases. ...
Article
Full-text available
Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA. Key Contribution: The reengineered Bacillus anthracis toxin has been applied to canine and human osteosarcoma cells, and inhibitory effects on cell viability and migration were observed in both cell types.
... With an estimated incidence of >10,000 new cases per year, osteosarcoma (OS) is the most common primary bone tumor in dogs. 1 Despite aggressive treatment involving limb amputation and systemic chemotherapy, 90% of dogs succumb to chemotherapeutic resistant metastatic disease. 2 Similar to canine OS, 30-40% of humans with OS die from metastatic disease despite treatment. [3][4][5] Targeted therapeutics and immunotherapy have revolutionized survival outcomes in a variety of cancers; however, to date, these have not translated in improved outcomes in either human or canine OS patients. [6][7][8][9][10][11][12][13] As systemic chemotherapy remains the backbone for treatment of OS metastases, the development of combinational treatments with novel and more effective agents is necessary for this disease. ...
Article
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Verdinexor (KPT‐335) is a novel orally bioavailable Selective Inhibitor of Nuclear Export (SINE) compound that inhibits the function of the nuclear export protein Exportin 1 (XPO1/CRM1). In the present study, we sought to characterize the expression of XPO1 in primary canine osteosarcoma (OS) tumor samples, OS cell lines and normal osteoblasts and evaluate the in vitro activity of verdinexor alone or in combination with doxorubicin. Canine OS cell lines and a subset of primary OS tumors showed increased XPO1 transcript and protein expression as compared to normal canine osteoblast cells. All canine OS cell lines exhibited dose‐dependent growth inhibition and increased caspase 3,7 activity in response to low nanomolar concentrations of verdinexor (IC50 concentrations ranging from 21‐74 nM). Notably, growth inhibition of normal canine osteoblast cell lines treated with verdinexor was observed at high micromolar concentrations (IC50 = 21 μM). The combination of verdinexor and doxorubicin resulted in potent inhibition of cell viability and demonstrated synergetic activity in three canine OS cell lines. Concordantly, OS cell lines showed increased γH2A.X foci following treatment with doxorubicin and recovery in verdinexor compared to cells treated with doxorubicin and recovered in normal media for 24 hours. These findings demonstrate that verdinexor has biologic activity against canine OS cell lines at physiologically relevant doses and suggest that XPO1 inhibition in combination with standard doxorubicin treatment offers promising potential for chemotherapeutic intervention in canine OS. This article is protected by copyright. All rights reserved.
Article
Osteosarcoma is an aggressive tumor of the bone that primarily affects young adults and adolescents. Osteosarcoma is characterized by genomic chaos and heterogeneity. While inactivation of tumor protein p53 (TP53) is nearly universal other high frequency mutations or structural variations have not been identified. Despite this genomic heterogeneity, key conserved transcriptional programs associated with survival have been identified across human, canine and induced murine osteosarcoma. The epigenomic landscape, including DNA methylation, plays a key role in establishing transcriptional programs in all cell types. The role of epigenetic dysregulation has been studied in a variety of cancers but has yet to be explored at scale in osteosarcoma. Here we examined genome-wide DNA methylation patterns in 24 human and 44 canine osteosarcoma samples identifying groups of highly correlated DNA methylation marks in human and canine osteosarcoma samples. We also link specific DNA methylation patterns to key transcriptional programs in both human and canine osteosarcoma. Building on previous work, we built a DNA methylation-based measure for the presence and abundance of various immune cell types in osteosarcoma. Finally, we determined that the underlying state of the tumor, and not changes in cell composition, were the main driver of differences in DNA methylation across the human and canine samples. Significance Genome wide comparison of DNA methylation patterns in osteosarcoma across two species lays the ground work for the exploration of DNA methylation programs that help establish conserved transcriptional programs in the context of varied mutational landscapes.
Chapter
To understand the pathophysiologic mechanisms of human bone and soft tissue sarcomas, and develop interventions to treat them, it became necessary to study sarcomas outside of the human body and deconstruct the events leading to full tumor formation. This has taken the form of human tumor-derived cell lines grown in culture, human tumor-derived cell lines and primary human tumors grown as xenografts in immunocompromised laboratory mice, genetically defined sarcoma cell lines, genetically engineered mouse models, and novel models of sarcoma arising spontaneously in domesticated animals or developed in lower organisms including zebrafish and fruit flies. This chapter reviews the uses of these approaches in understanding bone and soft tissue sarcomas and complements other recent sarcoma model reviews (O’Brien et al. 2012; Kashi et al. 2015).
Article
Background: Fine-needle aspirate (FNA) biopsy is considered a quick technique to access and identify the cell types present in a pathologic lesion or make a diagnosis. Often, clinicians want to know if they are dealing with an inflammatory lesion with or without infectious agents or a neoplastic lesion. At times, neoplastic lesions may be confounded by the presence of inflammatory cells. Objectives: We aimed to evaluate the application of a formalin-fixed FNA, designated the cytologic/histopathologic (CytoHisto) technique, to determine the diagnostic quality and ability to arrive at a definitive diagnosis without the use of concentrated cell block or invasive full tissue biopsy procedures during sample collection. Methods: A 10-cc syringe with a 22-gauge 1-inch needle attached was used to obtain a sizable FNA biopsy sample from a thigh mass in a dog. The needle was removed from the syringe, and the material was expulsed from the syringe directly into 10% buffered formalin. After 24-48-h fixation, a strainer was used to facilitate placement of the fixed granular material into a micromesh biopsy processing/embedding cassette, and the sample was processed as a routine histopathology sample. A microtome was used to make thin sections stained with H&E initially. Then, subsequent sections were stained with immunohistochemical (IHC) stains vimentin, MUM1, and CD18. Alkaline phosphatase staining was performed on a previously Wright's-stained cytology following IHC results. All sections were coverslipped and viewed under a light microscope. Results: When unable to perform incisional or excisional biopsies, this CytoHisto, FNA in formalin technique was useful for collection and subsequent processing as a histopathology sample, with sectioning and then staining with H&E and IHC stains. Neoplastic cells were strongly immunoreactive for vimentin but negative for MUM1 and CD18. Scattered leukocytes within the background stained positively with CD18. Conclusions: The CytoHisto technique is minimally invasive and allows for sectioning similar to a full-thickness excisional or incisional biopsy with subsequent H&E and IHC staining, and special stains allow for a definitive diagnosis of osteosarcoma. The CytoHisto technique is a practical diagnostic technique to pursue in clinical practice that minimizes patient invasiveness and maximizes sample collection time, similar to the routine FNA technique.
Article
Fluorine‐18‐fluorodeoxyglucose (18F‐FDG) positron emission tomography/computed tomography (PET/CT) has been utilized in veterinary medicine to improve the detection and characterization of primary, recurrent, and secondary neoplasms; but its use as a staging tool for dogs diagnosed with appendicular osteosarcoma has not been published. The purpose of this retrospective, case series, descriptive study was to detail the use of 18F‐FDG PET/CT for staging a population of dogs with appendicular osteosarcoma, report the detection rate of secondary neoplastic lesions, and compare findings with published detection rates for other historically used imaging modalities. Seventy‐one client‐owned dogs with a confirmed diagnosis of appendicular osteosarcoma and staged with a whole‐body 18F‐FDG PET/CT scan near the time of initial diagnosis were included. Each PET/CT study was re‐evaluated for malignancy distinct from the primary disease entity based on a collective qualitative and quantitative assessment of 18F‐FDG uptake, CT appearance, and contrast enhancement characteristics. Following re‐evaluation of each study, information pertaining to tissue sampling performed on identified lesions was retrieved from the medical record when available. Staging with 18F‐FDG PET/CT identified 17 of 71 (23.9%) and 12 of 71 (16.3%) dogs with a high suspicion or confirmation of a metastatic or comorbid malignant neoplasm respectively, with eight of 71 (11.3%) having both metastatic and comorbid lesions. The results of this study are suggestive that 18F‐FDG PET/CT is effective in identifying both osseous and soft tissue secondary neoplastic lesions in dogs afflicted with appendicular osteosarcoma, yielding an increased detection rate of all lesions compared those previously reported for skeletal scintigraphy or whole‐body CT.
Article
Periostin is a matricellular protein important in regulating bone, tooth, and cardiac development. In pathologic conditions, periostin drives allergic and fibrotic inflammatory diseases and is also overexpressed in certain cancers. Periostin signaling in tumors has been shown to promote angiogenesis, metastasis, and cancer stem cell survival in rodent models, and its overexpression is associated with poor prognosis in human glioblastoma. However, the role of periostin in regulating tumorigenesis of canine cancers has not been evaluated. Given its role in bone development, we sought to evaluate mRNA and protein expression of periostin in canine osteosarcoma (OS) and assess its association with patient outcome. We validated an anti-human periostin antibody cross-reactive to canine periostin via western blot and immunohistochemistry and evaluated periostin expression in microarray data from 49 primary canine OS tumors and 8 normal bone samples. Periostin mRNA was upregulated greater than 40-fold in canine OS tumors compared to normal bone and was significantly correlated with periostin protein expression based on quantitative image analysis. However, neither periostin mRNA nor protein expression were associated with time to metastasis in this cohort. Gene Set Enrichment Analysis demonstrated significant enhancement of pro-tumorigenic pathways including canonical WNT signaling, epithelial-mesenchymal transition, and angiogenesis in periostin-high tumors, while periostin-low tumors demonstrated evidence of heightened antitumor immune responses. Overall, these data identify a novel antibody that can be used as a tool for evaluation of periostin expression in dogs and suggest that investigation of Wnt pathway-targeted drugs in periostin overexpressing canine OS may be a potential therapeutic target.
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Purpose: The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically-achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression. Experimental design: 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a 2-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb followed by adjuvant carboplatin chemotherapy {plus minus} oral sirolimus therapy. The primary outcome measure was DFI, as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2- year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes. Results: There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days (95% CI: 144-237), and 282 days (95% CI: 224-383); for SOC + sirolimus dogs, it was 204 days (95% CI: 157-217) and 280 days (95% CI: 252-332), respectively. Conclusions: In a population of pet dogs non-genomically segmented for predicted mTOR inhibition response, sequentially-administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend disease-free interval or survival in dogs with appendicular osteosarcoma.
Article
Osteosarcoma (OSA) is the most common malignant bone cancer in dogs. Canine and human OSA share several features, including tumor environments, response to traditional treatment, and several molecular pathways. Hedgehog (Hh) signaling is known to contribute to tumorigenesis and progression of various cancers, including human OSA. This study aimed to identify the role of the Hh signaling pathway in canine OSA cell lines, including Abrams, D17, and Moresco, focusing on the signal transducer Smoothened (SMO). mRNA and protein levels of Hh pathway components, including SHH, IHH, SMO, and PTCH1, were aberrant in all examined OSA cell lines compared with canine osteoblast cells. The SMO inhibitor cyclopamine significantly decreased cell viability and colony‐forming ability in the canine OSA cell lines in a dose‐dependent manner. Moresco cells, which expressed the highest level of SMO protein, were the most sensitive to the anticancer effect of cyclopamine among the three canine OSA cell lines tested. Hh downstream target gene and protein expression in canine OSA cell lines were downregulated after cyclopamine treatment. In addition, cyclopamine significantly increased apoptotic cell death in Abrams and Moresco cells. The findings that Hh/SMO is activated in canine OSA cell lines and cyclopamine suppresses OSA cell survival via inhibition of SMO suggest that the Hh/SMO signaling pathway might be a novel therapeutic target for canine OSA. This article is protected by copyright. All rights reserved.
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Osteosarcoma is the most common bone tumor in children and young adults. Metastatic and relapsed disease confer poor prognosis, and there have been no improvements in outcomes for several decades. The disease's biological complexity, lack of drugs developed specifically for osteosarcoma, imperfect preclinical models, and limits of existing clinical trial designs have contributed to lack of progress. The Children's Oncology Group Bone Tumor Committee established the New Agents for Osteosarcoma Task Force to identify and prioritize agents for inclusion in clinical trials. The group identified multitargeted tyrosine kinase inhibitors, immunotherapies targeting B7‐H3, CD47‐SIRPα inhibitors, telaglenastat, and epigenetic modifiers as the top agents of interest. Only multitargeted tyrosine kinase inhibitors met all criteria for frontline evaluation and have already been incorporated into an upcoming phase III study concept. The task force will continue to reassess identified agents of interest as new data become available and evaluate novel agents using this method.
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Osteosarcoma has a guarded prognosis. A major hurdle in developing more effective osteosarcoma therapies is the lack of disease-specific biomarkers to predict risk, prognosis, or therapeutic response. Exosomes are secreted extracellular microvesicles emerging as powerful diagnostic tools. However, their clinical application is precluded by challenges in identifying disease-associated cargo from the vastly larger background of normal exosome cargo. We developed a method using canine osteosarcoma in mouse xenografts to distinguish tumor-derived from host-response exosomal messenger RNAs (mRNAs). The model allows for the identification of canine osteosarcoma-specific gene signatures by RNA sequencing and a species-differentiating bioinformatics pipeline. An osteosarcoma-associated signature consisting of five gene transcripts (SKA2, NEU1, PAF1, PSMG2, and NOB1) was validated in dogs with spontaneous osteosarcoma by real-time quantitative reverse transcription PCR (qRT-PCR), while a machine learning model assigned dogs into healthy or disease groups. Serum/plasma exosomes were isolated from 53 dogs in distinct clinical groups (“healthy”, “osteosarcoma”, “other bone tumor”, or “non-neoplastic disease”). Pre-treatment samples from osteosarcoma cases were used as the training set, and a validation set from post-treatment samples was used for testing, classifying as “osteosarcoma detected” or “osteosarcoma-NOT detected”. Dogs in a validation set whose post-treatment samples were classified as “osteosarcoma-NOT detected” had longer remissions, up to 15 months after treatment. In conclusion, we identified a gene signature predictive of molecular remissions with potential applications in the early detection and minimal residual disease settings. These results provide proof of concept for our discovery platform and its utilization in future studies to inform cancer risk, diagnosis, prognosis, and therapeutic response.
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Sažetak Liječenje tumorskih bolesti izuzetno je složen niz postupaka koji uključuje primjenu specifičnih lijekova, uglavnom kemoterapeutika, različitih postupaka fizičkog uništenja malignih novotvorevina poput zračenje, kirurških zahvata ili njihovih kombinacija. Opisani su kao agresivni za razliku od imunoterapije i virusne terapije te primjene različitih sredstava koja mogu na različite načine selektivno omogućiti propadanje tumorskih tkiva. Primjenom oba pristupa postiže se bolji učinak, manje oštećenje zdravog tkiva, a u većoj mjeri djeluje na tumorsko. Učinak pojedinih sredstava ili pristupa ocjenjuje se postupcima in vitro nakon čega se u istraživanj uključuju najprije male pokusne životinje, a zatim i psi. Genomski psi su vrlo slični čovjeku, a zbog životnog prostora što ga dijele, obolijevaju od niza bolesti kao i ljudi. Kraćeg životnog vijeka, u psa se bolesti općenito brže razvijaju pa tako predstavljaju idealnu pokusnu životinju u istraživanju različitih lijekova, posebice onih namijenjenih suzbijanju tumorskih bolesti. Psa kao pokusnu životinju iz etičkih se razloga ne bi smjelo koristiti, napose jer postoji mogućnosti njihova isključenja koje nam danas stoje na raspolaganju. Bez obzira na ovu činjenicu, psi i dalje obolijevaju od tumorskih bolesti. Upravo ti, bolesni psi bez izgleda za izliječenje, idealni su supstrat za primjenu sredstava različitih od onih agresivnih. Onkolitički virusi te imunoterapija u tom smislu, idealni su. Ponuđen je model po kojem i u nas, pas bi trebao predstavljati pacijenta a ne pokusnu životinju. Uključe li se institucije veterinarske znanosti i struke u ponuđni projekt, pas kao pacijent, poslužio bi u konačnici iznalaženju boljih postupaka sprječavanja pojave i liječenja tumorskih bolesti ljudi. Abstract The treatment of tumor diseases is an extremely complex series of procedures involving the use of specific drugs, mainly chemotherapeutics, various procedures for the physical destruction of malignant neoplasms such as radiation, surgery or a combination of these approaches. They have been described as aggressive in contrast to immunotherapy and viral therapy and the use of various agents that can selectively allow the deterioration of tumor tissues in various ways.
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In addition to their well-known functions in haemostasis, anucleated platelets have a critical role in cancer biology. Many human and non-human cancer types can directly interact with and activate platelets, promoting cancer malignancy and progression. Although naturally occurring canine neoplastic diseases mimic the biologically complex conditions of human cancers more closely than laboratory-bred mice, studies evaluating the relationship between cancer cells and platelets in dogs are scarce, and the effects of tumour cells on platelets in these animals are unknown. To evaluate whether cancer cells could activate canine platelets, we assessed the response of platelet-rich plasma to cultured canine cancer cells using light transmittance aggregometry. Similar to human and murine cancer cell research, we demonstrated that both canine osteosarcoma and mammary carcinoma cells activated canine platelets in vitro, resulting in platelet aggregation. The degree of aggregation was most pronounced at a cancer cell to platelet ratio of 1:200 for most cell lines. Mechanistic studies revealed that the platelet adenosine diphosphate (ADP) receptor P2Y12 is essential for canine platelet aggregation induced by canine cancer. ADP receptor blockage on platelets inhibited >50% of cancer cell-induced maximum platelet aggregation in all cell lines evaluated. As in other species, our results suggest that canine cancers may activate canine platelets in vivo. This mechanism is likely relevant for the biology and progression of cancer in the dog.
Article
Osteosarcoma (OS) is the most common malignant bone tumor in children. Despite efforts to develop and implement new therapies, patient outcomes have not measurably improved since the 1980s. Metastasis continues to be the main source of patient mortality, with 30% of cases developing metastatic disease within 5 years of diagnosis. Research models are critical in the advancement of cancer research and include a variety of species. For example, xenograft and patient-derived xenograft (PDX) mouse models provide opportunities to study human tumor cells in vivo while transgenic models have offered significant insight into the molecular mechanisms underlying OS development. A growing recognition of naturally occurring cancers in companion species has led to new insights into how veterinary patients can contribute to studies of cancer biology and drug development. The study of canine cases, including the use of diagnostic tissue archives and clinical trials, offers a potential mechanism to further canine and human cancer research. Advancement in the field of OS research requires continued development and appropriate use of animal models. In this review, animal models of OS are described with a focus on the mouse and tumor-bearing pet dog as parallel and complementary models of human OS.
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Canine osteosarcoma is clinically nearly identical to the human disease, but is common and highly heritable, making genetic dissection feasible. Through genome-wide association analyses in three breeds (greyhounds, Rottweilers, and Irish wolfhounds), we identify 33 inherited risk loci explaining 55% to 85% of phenotype variance in each breed. The greyhound locus exhibiting the strongest association, located 150 kilobases upstream of the genes CDKN2A/B, is also the most rearranged locus in canine osteosarcoma tumors. The top germline candidate variant is found at a >90% frequency in Rottweilers and Irish wolfhounds, and alters an evolutionarily constrained element that we show has strong enhancer activity in human osteosarcoma cells. In all three breeds, osteosarcoma-associated loci and regions of reduced heterozygosity are enriched for genes in pathways connected to bone differentiation and growth. Several pathways, including one of genes regulated by miR124, are also enriched for somatic copy-number changes in tumors. Mapping a complex cancer in multiple dog breeds reveals a polygenic spectrum of germline risk factors pointing to specific pathways as drivers of disease.
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Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma subtype, and fewer than half of patients are cured with standard front-line therapy. To improve therapeutic options, better animal models that accurately mimic human DLBCL (hDLBCL) are needed. Canine DLBCL (cDLBCL), one of the most common cancers in veterinary oncology, is morphologically similar to hDLBCL and is treated using similar chemotherapeutic protocols. With genomic technologies, it is now possible to molecularly evaluate dogs as a potential large-animal model for hDLBCL. We evaluated canine B-cell lymphomas (cBCLs) using immunohistochemistry and gene expression profiling. Canine B-cell lymphoma expression profiles were similar in many ways to hDLBCLs. For instance, a subset had increased expression of NF-κB pathway genes, mirroring human activated B-cell (ABC)-type DLBCL. Furthermore, immunoglobulin heavy chain (IGH) ongoing mutation status, which is correlated with ABC/germinal center B-cell (GCB) cell of origin in hDLBCL, separated cBCL into two groups with statistically different progression-free and overall survival times. In contrast with hDLBCL, cBCL rarely expressed BCL6 and MUM1/IRF4 by immunohistochemistry. Collectively, these studies identify molecular similarities to hDLBCL that introduce pet dogs as a representative model of hDLBCL for future studies, including therapeutic clinical trials.
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PURPOSEAberrant mammalian target of rapamycin (mTOR) signaling is common in sarcomas and other malignancies. Drug resistance and toxicities often limit benefits of systemic chemotherapy used to treat metastatic sarcomas. This large randomized placebo-controlled phase III trial evaluated the mTOR inhibitor ridaforolimus to assess maintenance of disease control in advanced sarcomas. PATIENTS AND METHODS Patients with metastatic soft tissue or bone sarcomas who achieved objective response or stable disease with prior chemotherapy were randomly assigned to receive ridaforolimus 40 mg or placebo once per day for 5 days every week. Primary end point was progression-free survival (PFS); secondary end points included overall survival (OS), best target lesion response, safety, and tolerability.ResultsA total of 711 patients were enrolled, and 702 received blinded study drug. Ridaforolimus treatment led to a modest, although significant, improvement in PFS per independent review compared with placebo (hazard ratio [HR], 0.72; 95% CI, 0.61 to 0.85; P = .001; median PFS, 17.7 v 14.6 weeks). Ridaforolimus induced a mean 1.3% decrease in target lesion size versus a 10.3% increase with placebo (P < .001). Median OS with ridaforolimus was 90.6 weeks versus 85.3 weeks with placebo (HR, 0.93; 95% CI, 0.78 to 1.12; P = .46). Adverse events (AEs) more common with ridaforolimus included stomatitis, infections, fatigue, thrombocytopenia, noninfectious pneumonitis, hyperglycemia, and rash. Grade ≥ 3 AEs were more common with ridaforolimus than placebo (64.1% v 25.6%). CONCLUSION Ridaforolimus delayed tumor progression to a small statistically significant degree in patients with metastatic sarcoma who experienced benefit with prior chemotherapy. Toxicities were observed with ridaforolimus, as expected with mTOR inhibition. These data provide a foundation on which to further improve control of sarcomas.
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Primary malignant bone tumors in the pediatric to young adult populations are relatively uncommon and account for about 6 % of all cancers in those less than 20 years old [1] and 3 % of all cancers in adolescents and young adults (AYA) within the age range of 15 to 29 years [2]. Osteosarcoma (OS) and Ewing's sarcoma (ES) comprise the majority of malignant bone tumors. The approach to treatment for both tumors consists of local control measures (surgery or radiation) as well as systemic therapy with high-dose chemotherapy. Despite earlier advances, there have been no substantial improvements in outcomes over the past several decades, particularly for patients with metastatic disease. This review summarizes the major advances in the treatment of OS and ES and the standard therapies available today, current active clinical trials, and areas of investigation into molecularly targeted therapies.
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Background High-grade osteosarcoma is an aggressive tumor most often developing in the long bones of adolescents, with a second peak in the 5th decade of life. Better knowledge on cellular signaling in this tumor may identify new possibilities for targeted treatment. Methods We performed gene set analysis on previously published genome-wide gene expression data of osteosarcoma cell lines (n=19) and pretreatment biopsies (n=84). We characterized overexpression of the insulin-like growth factor receptor (IGF1R) signaling pathways in human osteosarcoma as compared with osteoblasts and with the hypothesized progenitor cells of osteosarcoma – mesenchymal stem cells. This pathway plays a key role in the growth and development of bone. Since most profound differences in mRNA expression were found at and upstream of the receptor of this pathway, we set out to inhibit IR/IGF1R using OSI-906, a dual inhibitor for IR/IGF1R, on four osteosarcoma cell lines. Inhibitory effects of this drug were measured by Western blotting and cell proliferation assays. Results OSI-906 had a strong inhibitory effect on proliferation of 3 of 4 osteosarcoma cell lines, with IC50s below 100 nM at 72 hrs of treatment. Phosphorylation of IRS-1, a direct downstream target of IGF1R signaling, was inhibited in the responsive osteosarcoma cell lines. Conclusions This study provides an in vitro rationale for using IR/IGF1R inhibitors in preclinical studies of osteosarcoma.
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Background Deregulation of microRNA (miRNA) transcript levels has been observed in many types of tumors including osteosarcoma. Molecular pathways regulated by differentially expressed miRNAs may contribute to the heterogeneous tumor behaviors observed in naturally occurring cancers. Thus, tumor-associated miRNA expression may provide informative biomarkers for disease outcome and metastatic potential in osteosarcoma patients. We showed previously that clusters of miRNAs at the 14q32 locus are downregulated in human osteosarcoma. Methods Human and canine osteosarcoma patient’s samples with clinical follow-up data were used in this study. We used bioinformatics and comparative genomics approaches to identify miRNA based prognostic biomarkers in osteosarcoma. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. Results Here we show that an inverse correlation exists between aggressive tumor behavior (increased metastatic potential and accelerated time to death) and the residual expression of 14q32 miRNAs (using miR-382 as a representative of 14q32 miRNAs) in a series of clinically annotated samples from human osteosarcoma patients. We also show a comparable decrease in expression of orthologous 14q32 miRNAs in canine osteosarcoma samples, with conservation of the inverse correlation between aggressive behavior and expression of orthologous miRNA miR-134 and miR-544. Conclusions We conclude that downregulation of 14q32 miRNA expression is an evolutionarily conserved mechanism that contributes to the biological behavior of osteosarcoma, and that quantification of representative transcripts from this family, such as miR-382, miR-134, and miR-544, provide prognostic and predictive markers that can assist in the management of patients with this disease.
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Background STAT3 [1] has been shown to be dysregulated in nearly every major cancer, including osteosarcoma (OS). Constitutive activation of STAT3, via aberrant phosphorylation, leads to proliferation, cell survival and resistance to apoptosis. The present study sought to characterize the biologic activity of a novel allosteric STAT3 inhibitor, LLL12, in canine OS cell lines. Results We evaluated the effects of LLL12 treatment on 4 canine OS cell lines and found that LLL12 inhibited proliferation, induced apoptosis, reduced STAT3 phosphorylation, and decreased the expression of several transcriptional targets of STAT3 in these cells. Lastly, LLL12 exhibited synergistic anti-proliferative activity with the chemotherapeutic doxorubicin in the OS lines. Conclusion LLL12 exhibits biologic activity against canine OS cell lines through inhibition of STAT3 related cellular functions supporting its potential use as a novel therapy for OS.
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Here we report a high-quality draft genome sequence of the domestic dog (Canis familiaris), together with a dense map of single nucleotide polymorphisms (SNPs) across breeds. The dog is of particular interest because it provides important evolutionary information and because existing breeds show great phenotypic diversity for morphological, physiological and behavioural traits. We use sequence comparison with the primate and rodent lineages to shed light on the structure and evolution of genomes and genes. Notably, the majority of the most highly conserved non-coding sequences in mammalian genomes are clustered near a small subset of genes with important roles in development. Analysis of SNPs reveals long-range haplotypes across the entire dog genome, and defines the nature of genetic diversity within and across breeds. The current SNP map now makes it possible for genome-wide association studies to identify genes responsible for diseases and traits, with important consequences for human and companion animal health.
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Background Myeloid derived suppressor cells (MDSCs) are a recently described population of immune cells that significantly contribute to the immunosuppression seen in cancer patients. MDSCs are one of the most important factors that limit the efficacy of cancer immunotherapy (e.g. cancer vaccines) and MDSC levels are increased in cancer in multiple species. Identifying and targeting MDSCs is actively being investigated in the field of human oncology and is increasingly being investigated in veterinary oncology. The treatment of canine cancer not only benefits dogs, but is being used for translational studies evaluating and modifcying candidate therapies for use in humans. Thus, it is necessary to understand the immune alterations seen in canine cancer patients which, to date, have been relatively limited. This study investigates the use of commercially available canine antibodies to detect an immunosuppressive (CD11blow/CADO48low) cell population that is increased in the peripheral blood of tumor-bearing dogs. Results Commercially available canine antibodies CD11b and CADO48A were used to evaluate white blood cells from the peripheral blood cells of forty healthy control dogs and forty untreated, tumor-bearing dogs. Tumor-bearing dogs had a statistically significant increase in CD11blow/CADO48Alow cells (7.9%) as compared to the control dogs (3.6%). Additionally, sorted CD11blow/CADO48Alow generated in vitro suppressed the proliferation of canine lymphocytes. Conclusions The purpose of this study was aimed at identifying potential canine specific markers for identifying MDSCs in the peripheral blood circulation of dogs. This study demonstrates an increase in a unique CD11blow/CADO48Alow cell population in tumor-bearing dogs. This immunophenotype is consistent with described phenotypes of MDSCs in other species (i.e. mice) and utilizes commercially available canine-specific antibodies. Importantly, CD11blow/CADO48Alow from a tumor environment suppress the proliferation of lymphocytes. These results provide a useful phenotype of cells increased in canine cancer patients that may serve as a useful prognostic marker for assessing immune status and functional response to cancer immunotherapies in dogs. Understanding MDSCs in dogs will allow for increased effectiveness of cancer immunotherapy in both dogs and humans.
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Canine osteosarcoma (OSA) is known to present with highly variable and chaotic karyotypes, including hypodiploidy, hyperdiploidy, and increased numbers of metacentric chromosomes. The spectrum of genomic instabilities in canine OSA has significantly augmented the difficulty in clearly defining the biological and clinical significance of the observed cytogenetic abnormalities. In this study, eight canine OSA cell lines were used to investigate telomere fusions by fluorescence in situ hybridization (FISH) using a peptide nucleotide acid probe. We characterized each cell line by classical cytogenetic studies and cellular phenotypes including telomere associated factors and then evaluated correlations from this data. All eight canine OSA cell lines displayed increased abnormal metacentric chromosomes and exhibited numerous telomere fusions and interstitial telomeric signals. Also, as evidence of unstable telomeres, colocalization of γ-H2AX and telomere signals in interphase cells was observed. Each cell line was characterized by a combination of data representing cellular doubling time, DNA content, chromosome number, metacentric chromosome frequency, telomere signal level, cellular radiosensitivity, and DNA-PKcs protein expression level. We have also studied primary cultures from 10 spontaneous canine OSAs. Based on the observation of telomere aberrations in those primary cell cultures, we are reasonably certain that our observations in cell lines are not an artifact of prolonged culture. A correlation between telomere fusions and the other characteristics analyzed in our study could not be identified. However, it is important to note that all of the canine OSA samples exhibiting telomere fusion utilized in our study were telomerase positive. Pending further research regarding telomerase negative canine OSA cell lines, our findings may suggest telomere fusions can potentially serve as a novel marker for canine OSA.
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Fas ligand (FasL) gene therapy for cancer has shown promise in rodents; however, its efficacy in higher mammals remains unknown. Here, we used intratumoral FasL gene therapy delivered in an adenovirus vector (Ad-FasL) as neoadjuvant to standard of care in 56 dogs with osteosarcoma. Tumors from treated dogs had greater inflammation, necrosis, apoptosis, and fibrosis at day 10 (amputation) compared to pretreatment biopsies or to tumors from dogs that did not receive Ad-FasL. Survival improvement was apparent in dogs with inflammation or lymphocyte-infiltration scores >1 (in a 3-point scale), as well as in dogs that had apoptosis scores in the top 50th percentile (determined by cleaved caspase-3). Survival was no different than that expected from standard of care alone in dogs with inflammation scores ≤1 or apoptosis scores in the bottom 50th percentile. Reduced Fas expression by tumor cells was associated with prognostically advantageous inflammation, and this was seen only in dogs that received Ad-FasL. Together, the data suggest that Ad-FasL gene therapy improves survival in a subset of large animals with naturally occurring tumors, and that at least in some tumor types like osteosarcoma, it is most effective when tumor cells fail to express Fas.
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Background: This study was designed to assess the efficacy of a matrix metalloproteinase inhibitor in prolonging posttreatment survival for dogs with appendicular osteosarcoma after treatment with amputation and doxorubicin chemotherapy.Hypothesis: Survival will be prolonged in dogs receiving BAY 12–9566.Animals: The study included 303 dogs with appendicular osteosarcoma.Methods: Dogs were treated with doxorubicin (30 mg/m2) every 2 weeks for 5 treatments starting 2 weeks after amputation. Dogs were randomly allocated to receive a novel nonpeptidic biphenyl inhibitor of matrix metalloproteinases (MMPs, BAY 12–9566; 4-[4–4-(chlorophenyl)phenyl]-4-oxo-2S-(phenylthiomethyl) butanoic acid) or placebo after doxorubicin chemotherapy.Results: Median survival for all 303 dogs was 8 months; and 1-year, 2-year, and 3-year survival rates were 35%, 17%, and 9%, respectively. Treatment with BAY 12–9566 did not influence survival. Multivariate analysis revealed that increasing age (P= .004), increasing weight (P= .006), high serum alkaline phosphatase (ALP) (P= .012) and high bone ALP (P < .001) were independently associated with shorter median survival times. Additional analyses on available data indicated that as the number of mitotic figures in the biopsy increased (P= .013), and as plasma active MMP-2 concentrations increased (P= .027), the risk of dying increased.Conclusions and Clinical Importance: Doxorubicin is an effective adjuvant to amputation in prolonging survival for dogs with appendicular osteosarcoma.
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Background Appendicular osteosarcoma is the most common malignant primary canine bone tumor. When treated by amputation or tumor removal alone, median survival times (MST) do not exceed 5 months, with the majority of dogs suffering from metastatic disease. This period can be extended with adequate local intervention and adjuvant chemotherapy, which has become common practice. Several prognostic factors have been reported in many different studies, e.g. age, breed, weight, sex, neuter status, location of tumor, serum alkaline phosphatase (SALP), bone alkaline phosphatase (BALP), infection, percentage of bone length affected, histological grade or histological subtype of tumor. Most of these factors are, however, only reported as confounding factors in larger studies. Insight in truly significant prognostic factors at time of diagnosis may contribute to tailoring adjuvant therapy for individual dogs suffering from osteosarcoma. The objective of this study was to systematically review the prognostic factors that are described for canine appendicular osteosarcoma and validate their scientific importance. Results A literature review was performed on selected studies and eligible data were extracted. Meta-analyses were done for two of the three selected possible prognostic factors (SALP and location), looking at both survival time (ST) and disease free interval (DFI). The third factor (age) was studied in a qualitative manner. Both elevated SALP level and the (proximal) humerus as location of the primary tumor are significant negative prognostic factors for both ST and DFI in dogs with appendicular osteosarcoma. Increasing age was associated with shorter ST and DFI, however, was not statistically significant because information of this factor was available in only a limited number of papers. Conclusions Elevated SALP and proximal humeral location are significant negative prognosticators for canine osteosarcoma.
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