Reason for euthanasia in dogs with urothelial carcinoma treated with chemotherapy or radiation therapy or both: A retrospective observational study.

BACKGROUND: Clients want to know the ultimate cause of death in their pet after cancer treatment. The cause of euthanasia and investigation of urinary obstruction in treated dogs with urothelial carcinoma (UC) has not been specifically reported in veterinary literature. HYPOTHESIS/OBJECTIVES: Our hypothesis was that the majority of treated dogs with UC are euthanized secondary to primary tumor factors, such as urinary obstruction. ANIMALS: Fifty-nine client-owned dogs diagnosed with UC. METHODS: Retrospective observational study on clinical signs and disease at euthanasia of dogs with UC treated by radiation therapy or chemotherapy or both. RESULTS: The median overall survival time (OST) of all dogs was 339 days (range, 17-1996; 95% confidence interval [CI], 185-392; interquartile range [IQR], 112-505). Of dogs deemed to have been euthanized because of UC (50/59, 85%), the primary cause was considered to be local progression in 31/50 (62%), most often because of perceived complete or partial urinary obstruction (24/31, 77%). No variables were found to be predictive of urinary obstruction. The overall documented metastatic rate was 56%. In dogs euthanized because of UC, metastasis was deemed to be the cause in 19/50 (38%) dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: Regardless of the type of treatment, UC in dogs has a poor prognosis and there is a continuing need to improve treatments that focus on local control of the primary tumor, given its high contribution to the decision for euthanasia. Proactive management to avoid the high frequency of urinary obstruction may be worthy of future investigation.

Quantification of circulating tumour cells over time in dogs with appendicular osteosarcoma.

Enumeration of circulating tumour cells (CTC) has shown promise for prognostication and guidance of therapeutic decisions in human cancers. The objective of this study was to enumerate CTC over time in dogs with naturally occurring osteosarcoma (OSA), and to determine correlation with patient outcome. Twenty-six dogs with OSA and no evidence of metastatic disease at the time of amputation were enrolled. Dogs were assessed for lung metastases and CTC prior to and following amputation, and at each chemotherapy visit. Twenty-one dogs completed the study. Nineteen dogs were euthanized and two were alive and free of metastases. Overall survival time ranged from 88 to 1058 days (median survival time (MST) 374 days). Increased serum alkaline phosphatase activity, advanced age, and higher body weight were significantly associated with lower MST. Dogs with OSA had a mean of 356 (0 to 4443) CTC/106 leukocytes. In 12 of 15 dogs that developed radiographic evidence of metastasis, a pre-metastatic CTC spike was retrospectively detectable on average 36.5 (1-100 days) days prior to metastasis and was associated with significantly shorter MST (301 ± 64 vs. 626 ± 55 days; p = .0107). In a multivariable analysis, dogs with a CTC spike were 10× more likely to die compared with those without. These results suggest that a spike in CTC frequency precedes detection of metastasis in dogs with OSA and is associated with shorter survival. More frequent enumeration of CTC in a larger cohort of dogs with OSA may be warranted.

Treatment of myeloid neoplasia with doxorubicin and cytarabine in 11 dogs.

Myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML) are primary myeloid neoplasms in dogs generally considered to have a poor outcome. In this study, we assessed toxicity, efficacy and outcome of concurrent administration of doxorubicin and cytarabine in 11 dogs with myeloid neoplasia. Bone marrow specimens were reviewed by three pathologists and classified as either MDS (n = 2), high grade MDS/early AML (MDS/AML; n = 4) or AML (n = 5). The median number of treatment cycles was 5 (range 1-9) and resolution of cytopenia was reported in 7 of 11 dogs including 2 dogs with MDS, 2 dogs with MDS/AML, and 3 dogs with AML. The median duration of remission in the seven responders was 344 days (range 109-1428) and the median overall survival for all dogs was 369 days. Adverse events consisted of predominantly low-grade gastrointestinal illness and myelosuppression. Three dogs developed grade V toxicity manifesting with heart failure (n = 2) at 369 and 1170 days after diagnosis and acute gastrointestinal side effects (n =1). Despite a limited sample size, these results suggest that a doxorubicin and cytarabine protocol may be considered as a therapeutic option in dogs with myeloid neoplasia. Protocol safety, in particular regarding myocardial toxicity, and efficacy should be further investigated.

Prognostic significance of the urokinase plasminogen activator system in tissue and serum of dogs with appendicular osteosarcoma.

Urokinase plasminogen activator (uPA) and its receptor uPAR promote cancer invasion and metastasis and are emerging therapeutic targets in both human and canine malignancies. While their clinical significance is well-characterized in multiple human tumor types, studies investigating their roles in osteosarcoma are lacking. The objectives of this study were to characterize serum and tissue uPA/uPAR expression in dogs with osteosarcoma and assess the prognostic significance. Serum samples and a tissue microarray of canine appendicular osteosarcoma were analyzed for uPA and uPAR expression by ELISA (n = 49) and immunohistochemistry (n = 38), respectively. Serum uPA activity was also measured by a chromogenic assay (n = 25). Survival analysis was performed by Kaplan-Meier survival analysis, log rank test, and Cox regression analysis. Serum uPA level was significantly higher in dogs with osteosarcoma than clinically healthy control dogs (median 1905 vs 1440 pg/ml, p = 0.008). The majority of canine osteosarcoma tissues expressed uPA (75.9%) or uPAR (77.6%), with 70.7% dual-positivity, indicating autocrine/paracrine activation of the pathway. Survival analysis revealed shorter progression free survival (PFS) in dogs with high serum uPA level in a discovery cohort (n = 29; median PFS 94 vs 266 days, p = 0.003) but not in a validation cohort (n = 23; median PFS 167 vs 490 days, p = 0.16). The difference was significant when both cohorts were combined (n = 49; median PFS 128 vs 266 days, p = 0.003). Serum uPAR and tissue uPA/uPAR levels were not prognostic. In Cox multivariate analysis, high serum uPA level and activity were both associated with poor prognosis, independent of serum ALP, tumor location, and peripheral lymphocyte/monocyte counts. These results indicate high utilization of the uPA pathway and association with disease progression in canine osteosarcoma. Further study involving prospective evaluation to confirm the prognostic significance is warranted. The high prevalence of tissue uPA and uPAR expression suggests the uPA system as a potential therapeutic target in canine osteosarcoma.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Evaluation of PD-1 and PD-L1 expression in canine urothelial carcinoma cell lines.

Urothelial carcinoma (UC) is the most common urinary tumor in dogs and despite combinational therapies, only modest improvements in survival have been achieved in recent years. Given the utility of monoclonal antibodies against PD-1 and PD-L1 in human UC, we evaluated the protein and mRNA expression in three established canine urothelial carcinoma cell lines. Flow cytometry and western blot analysis confirmed cell line expression of both molecules in varying degrees. Reverse transcription PCR (RT-PCR) documented mRNA expression in all three cell lines for both PD-1 and PD-L1. Fluorescence microscopy was consistent with strong PD-1 and PD-L1 expression in the canine cell lines and was in line with previous human literature. Importantly, the flow cytometry work described in this study revealed higher cell intrinsic PD-1 expression in these cell lines which may have implications for tumor behavior and potential treatment opportunities in the future. Further work is necessary to determine the expression patterns in canine UC and potential for benefit with immunotherapy directed against PD-1 and PD-L1.

Evaluation of lymphocyte-specific programmed cell death protein 1 receptor expression and cytokines in blood and urine in canine urothelial carcinoma patients.

Urothelial carcinoma (UC) is the most common urinary tumour in dogs. Despite a range of treatment options, prognosis remains poor in dogs. In people, breakthroughs with checkpoint inhibitors have established new standards of care for muscle-invasive bladder cancer patients and elevated levels of programmed cell death protein 1 (PD-1) suggest immune checkpoint blockade may be a novel target for therapy. The goal of this study was to determine if canine UC patients express elevated levels of lymphocyte-specific PD-1 and/or urinary cytokine biomarkers compared to healthy dogs. Paired blood and urine were evaluated in 10 canine UC patients, five cystitis patients and 10 control dogs for lymphocyte-specific PD-1 expression via flow cytometry and relative cytokine expression. In UC patients, PD-1 expression was significantly elevated on CD8+ lymphocytes in urine samples. UC patients had a higher CD4:CD8 ratio in their urine compared to healthy dogs, however, there was no significant variation in the CD8:Treg ratio between any group. Cystitis patients had significantly elevated levels of CD4+ T cells, CD8+ T cells and Tregs in their blood samples compared to UC patients and healthy dogs. Cytokine analysis demonstrated significant elevations in urinary cytokines (granulocyte-macrophage colony-stimulating factor, interferon-gamma [IFN-γ], interleukin (IL)-2, IL-6 IL-7, IL-8 and IL-15, IP-10, KC-like, IL-18, monocyte chemoattractant protein-1 and tumour necrosis factor-alpha). Several of these cytokines have been previously correlated with both lymphocyte-specific PD-1 expression (IFN-γ, IL-2, IL-7 and IL-15) in muscle-invasive urothelial carcinoma in humans. Our results provide evidence of urinary lymphocyte PD-1 expression and future studies could elucidate whether veterinary UC patients will respond favourably to anti-PD-1 immune checkpoint inhibitor therapy.

Using a Prime-Boost Vaccination Strategy That Proved Effective for High Resolution Epitope Mapping to Characterize the Elusive Immunogenicity of Survivin.

Survivin is a member of the inhibitor of apoptosis family of proteins and has been reported to be highly expressed in a variety of cancer types, making it a high priority target for cancer vaccination. We previously described a heterologous prime-boost strategy using a replication-deficient adenovirus, followed by an oncolytic rhabdovirus that generates unprecedented antigen-specific T cell responses. We engineered each vector to express a mutated version of full-length murine survivin. We first sought to uncover the complete epitope map for survivin-specific T cell responses in C57BL/6 and BALB/c mice by flow cytometry. However, no T cell responses were detected by intracellular cytokine staining after re-stimulation of T cells. Survivin has been found to be expressed by activated T cells, which could theoretically cause T cell-mediated killing of activated T cells, known as fratricide. We were unable to recapitulate this phenomenon in experiments. Interestingly, the inactivated survivin construct has been previously shown to directly kill tumor cells in vitro. However, there was no evidence in our models of induction of death in antigen-presenting cells due to treatment with a survivin-expressing vector. Using the same recombinant virus-vectored prime-boost strategy targeting the poorly immunogenic enhanced green fluorescent protein proved to be a highly sensitive method for mapping T cell epitopes, particularly in the context of identifying novel epitopes recognized by CD4+ T cells. Overall, these results suggested there may be unusually robust tolerance to survivin in commonly used mouse strains that cannot be broken, even when using a particularly potent vaccination platform. However, the vaccination method shows great promise as a strategy for identifying novel and subdominant T cell epitopes.

Investigation of the effects of mTOR inhibitors rapamycin and everolimus in combination with carboplatin on canine malignant melanoma cells.

BACKGROUND: Malignant melanoma in dogs is considered to be largely resistant to conventional chemotherapy, although responses to carboplatin have been documented. Invasion and early metastasis are common features of certain melanoma subtypes that contribute to tumour progression despite aggressive local and systemic therapy. Upregulation of the PI3K/AKT/mTOR pathway has been observed in canine malignant melanoma and may represent a potential target for therapy. Rapamycin (sirolimus) and everolimus are commercially available small molecule inhibitors that target mTOR and therefore may have anticancer activity in canine melanoma. It was hypothesized that there is synergism between rapamycin or everolimus and platinum chemotherapy, and that combination drug treatment would inhibit target/downstream proteins involved in cell viability/proliferation and increase cell death in canine melanoma cells. It was further hypothesized that rapamycin or everolimus would impact metabolism by reducing glycolysis in these cells. Four canine melanoma cell lines were treated in vitro with rapamycin and everolimus as sole treatment or combined with carboplatin. Cell viability, apoptosis, target modulation, and glycolytic metabolism were evaluated by crystal violet colourimetric assay, Annexin V/PI flow cytometry, western blotting, and Seahorse bioanalyzer, respectively. RESULTS: When combined with carboplatin chemotherapy, rapamycin or everolimus treatment was overall synergistic in reducing cell viability. Carboplatin-induced apoptosis was noted at 72 h after treatment compared to the vehicle control. Levels of phosphorylated mTOR were reduced by rapamycin and everolimus in all four cell lines, but activation of the downstream protein p70S6K was not consistently reduced by treatment in two of the cell lines. Both mTOR inhibitors decreased the extracellular acidification rate of canine melanoma cells, indicating reduced cancer cell glycolytic activity. CONCLUSIONS: Inhibition of mTOR by rapalogs, such as rapamycin and everolimus combined with carboplatin chemotherapy may have activity in canine melanoma. Future mechanistic investigation is warranted, including in vivo assessment of this combination therapy.

Combination therapy with cannabidiol and chemotherapeutics in canine urothelial carcinoma cells.

BACKGROUND: Canine urothelial carcinoma is the most common form of canine bladder cancer. Treatment with chemotherapy has variable response rates leading to most dogs succumbing to their disease within a year. Cannabidiol is an emerging treatment within the field of oncology. In reported in vivo studies, cannabidiol has induced apoptosis, reduced cell migration, and acted as a chemotherapy sensitizer in various human tumor types. The aim of this study was to characterize the effects of cannabidiol on canine urothelial carcinoma cell viability and apoptosis as both a single agent and in combination with chemotherapy in vitro. RESULTS: Cannabidiol reduced cell viability and induced apoptosis in canine urothelial cells as determined by crystal violet viability assay and annexin V/propidium iodide flow cytometry. Furthermore, combinations of cannabidiol with mitoxantrone and vinblastine chemotherapy yielded significantly reduced cell viability and increased apoptosis compared to single agent treatment alone. The drug interactions were deemed synergistic based on combination index calculations. Conversely, the combination of cannabidiol and carboplatin did not result in decreased cell viability and increased apoptosis compared to single agent treatment. Combination index calculations suggested an antagonistic interaction between these drugs. Finally, the combination of the non-steroidal anti-inflammatory drug piroxicam with cannabidiol did not significantly affect cell viability, although, some cell lines demonstrated decreased cell viability when mitoxantrone was combined with piroxicam. CONCLUSIONS: Cannabidiol showed promising results as a single agent or in combination with mitoxantrone and vinblastine for treatment of canine urothelial carcinoma cells. Further studies are justified to investigate whether these results are translatable in vivo.

Adjuvant Sirolimus Does Not Improve Outcome in Pet Dogs Receiving Standard-of-Care Therapy for Appendicular Osteosarcoma: A Prospective, Randomized Trial of 324 Dogs.

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. PATIENTS AND METHODS: A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (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% confidence interval (CI), 144-237] and 282 days (95% CI, 224-383) and 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 nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

Plasma 25-hydroxyvitamin D and the inflammatory response in canine cancer.

Decreased circulating 25-hydroxyvitamin D (25[OH]D) and increased inflammatory marker concentrations have been reported separately in canine cancer. Correlations between the two exist in humans, but little work has examined links in dogs. This study aimed to determine plasma 25(OH)D and inflammatory marker concentrations in healthy dogs and dogs with cancer and to assess correlations in each group. Newly diagnosed dogs with B-cell lymphoma (B-cell, n = 25), T-cell lymphoma (T-cell, n = 9), osteosarcoma (OSA, n = 21), and mast cell tumour (MCT, n = 26) presenting to a tertiary oncology centre, and healthy dogs (n = 25), were enrolled. Plasma samples were analysed for 25(OH)D, C-reactive protein (CRP), haptoglobin (HP), serum amyloid A (SAA), alpha-1-acid glycoprotein (AAG), and 13 chemokines and cytokines. Dogs with B-cell had decreased plasma 25(OH)D (P = .03), and increased plasma CRP, AAG, HP, KC-like and MCP-1 concentrations (P < =.001, .011, <.001, .013 and .009, respectively) compared with healthy dogs. Plasma CRP, HP and SAA concentrations were increased in dogs with OSA compared with healthy dogs (P = .001, .010 and .027, respectively). No differences were noted in dogs with T-cell and MCT. Negative correlations were observed between plasma 25(OH)D concentrations and: AAG concentrations in dogs with T-cell (Rs  = -0.817, P = .007); GM-CSF concentrations (Rs  = -0.569, P = .007) in dogs with OSA; and IL-7 concentrations (Rs  = -0.548, P = .010) in dogs with OSA. Decreased 25(OH)D concentrations and increased concentrations of multiple inflammatory markers were observed in B-cell patients, supporting an association between 25(OH)D and inflammation. The cross-sectional study design meant the timing of changes could not be determined. Prospective cohort studies are warranted.

Effect of timing of bisphosphonate administration on canine osteosarcoma cells undergoing radiation therapy.

The effects of radiation therapy may be potentiated by combining radiation therapy with secondary therapies. Clinically, radiation therapy has been combined with bisphosphonates for treatment of canine appendicular osteosarcoma for years. The objective of this study was to determine if the timing of administration of bisphosphonates in relation to radiation therapy alters clonogenic survival or cell viability of canine osteosarcoma cells in vitro. Canine osteosarcoma cells were treated before administration of radiation, concurrent with radiation, or after radiation. Reduction in clonogenic survival was identified when bisphosphonates were administered post-radiation compared with pre-radiation. No significant differences were identified for cell viability at any time points. Further investigation of the cellular effects of bisphosphonates on canine osteosarcoma cells is warranted. Consideration may be given to administering bisphosphonates 24 h after radiation to reduce replication of canine osteosarcoma cells and possibly prolong the analgesic effects of both treatments.

Les effets de la radiothérapie peuvent être potentialisés en associant la radiothérapie à des thérapies secondaires. Cliniquement, la radiothérapie est associée aux bisphosphonates pour le traitement de l'ostéosarcome appendiculaire canin depuis des années. L'objectif de cette étude était de déterminer si le moment choisi pour l'administration de bisphosphonates en relation avec la radiothérapie altère la survie clonogénique ou la viabilité cellulaire de cellules d'ostéosarcome canin in vitro. Les cellules d'ostéosarcome canin ont été traitées avant l'administration d'un rayonnement, concomitant avec le rayonnement ou après l'administration d'un rayonnement. La réduction de la survie clonogénique a été identifiée lorsque les bisphosphonates étaient administrés post-irradiation par rapport à l'irradiation. Aucune différence significative n'a été identifiée pour la viabilité des cellules à aucun moment. Une étude plus approfondie des effets cellulaires des bisphosphonates sur les cellules d'ostéosarcome canin est justifiée. L'administration de bisphosphonates peut être envisagée 24 heures après l'administration de radiations afin de réduire la réplication des cellules d'ostéosarcome canin.(Traduit par les auteurs).

Recent and current clinical trials in canine appendicular osteosarcoma.

Osteosarcoma (OSA) is an aggressive primary bone tumor in the domestic dog that most often occurs within the appendicular skeleton. Despite the use of adjuvant chemotherapy, most dogs succumb to metastatic disease within 1 year of diagnosis. To improve this outcome, substantial research is currently focused on investigating novel therapies. Herein, we review emerging treatments and clinical trials that, if proven efficacious, could revolutionize the standard of care for canine appendicular OSA. This article includes a critical perspective on the safety, efficacy, and limitations of select immunotherapy, virotherapy, radiotherapy, targeted therapy, and personalized medicine trials, all of which reflect similar investigations taking place in human oncology. These clinical trials represent a major evolution in the overall approach to therapy for dogs with appendicular OSA that could have significant implications for improving survival.

Essais cliniques récents et en cours sur l'ostéosarcome appendiculaire canin. L'ostéosarcome (OSA) est une tumeur osseuse primaire agressive chez le chien domestique qui se produit fréquemment dans le squelette appendiculaire. Malgré l'utilisation de chimiothérapie complémentaire, la majorité des chiens succombent aux métastases en dedans d'une année du diagnostic. Afin d'améliorer ce résultat, de la recherche substantielle est actuellement concentrée sur l'étude de thérapies nouvelles. À cet égard, nous révisons les traitements émergents et les essais cliniques qui, s'ils s'avèrent efficaces, pourraient révolutionner le standard de soins pour les OSA appendiculaires canins. Le présent article inclus une perspective critique de la sécurité, l'efficacité et les limitations d'un choix d'immunothérapie, de virothérapie, de radiothérapie, de thérapies ciblées et d'essais médicaux personnalisés, qui reflètent tous des investigations similaires effectuées en oncologie humaine. Ces essais cliniques représentent une évolution majeure dans l'approche globale à la thérapie de chiens avec OSA appendiculaire qui pourrait avoir des implications significatives pour améliorer la survie.(Traduit par Dr Serge Messier).

Inhibition of copper chaperones sensitizes human and canine osteosarcoma cells to carboplatin chemotherapy.

Osteosarcoma (OSA) is the most common primary bone cancer in children, adolescents and dogs. Current combination surgical and chemotherapeutic treatments have increased survival. However, in recurrent or metastatic disease settings, the prognosis significantly decreases, representing an urgent need for better second-line and novel chemotherapeutics. The current gold standard for combination chemotherapy in OSA often includes a platinum agent, for example, cisplatin or carboplatin. These platinum agents are shuttled within the cell via copper transporters. Recent interest in targeting copper transport has been directed towards antioxidant protein 1 (Atox1) and copper chaperone for superoxide dismutase 1 (CCS), with Atox1 demonstrating the ability to aggregate platinum agents, preventing them from forming DNA adducts. DC_AC50 is a small molecule inhibitor of both Atox1 and CCS. To assess the impact of targeting these pathways on chemotherapy response, two human and two canine OSA cell lines were utilized. After treatment with single agent or combination drugs, cell viability was evaluated and pharmacological synergism calculated using the combination index method. Apoptosis, cell cycle distribution, clonogenic survival and migration were also evaluated. DC_AC50 synergised with carboplatin in combination treatment of human and canine OSA cells to reduce cancer cell viability. DC_AC50-treated cells were significantly less mitotically active, as demonstrated by decreased expression of phospho-histone H3 and cell cycle analysis. DC_AC50 also potentiated carboplatin-induced apoptosis in OSA cells and decreased clonogenic survival. Finally, DC_AC50 reduced the migratory ability of OSA cells. These results justify further investigation into inhibiting intracellular copper chaperones as a means of reducing/preventing acquired chemotherapy resistance.

MicroRNA profiling in canine multicentric lymphoma.

Lymphoma is the most common hematopoietic tumour in dogs and is remarkably similar to the human disease. Tumour biomarker discovery is providing new tools for diagnostics and predicting therapeutic response and clinical outcome. MicroRNAs are small non-coding RNAs that participate in post-transcriptional gene regulation and their aberrant expression can impact genes involved in cancer. The aim of this study was to characterize microRNA expression in lymph nodes and plasma from dogs with multicentric B or T cell lymphoma compared to healthy control dogs. We further compared expression between lymph nodes and corresponding plasma samples and assessed changes in expression at relapse compared to time of diagnosis. Lastly, we investigated microRNAs for association with clinical outcome in patients treated with CHOP chemotherapy. A customized PCR array was utilized to profile 38 canine target microRNAs. Quantification was performed using real time RT-qPCR and relative expression was determined by the delta-delta Ct method. In lymph nodes, there were 16 microRNAs with significantly altered expression for B cell lymphoma and 9 for T cell lymphoma. In plasma, there were 15 microRNAs altered for B cell lymphoma and 3 for T cell lymphoma. The majority of microRNAs did not have correlated expression between lymph node and plasma and only 8 microRNAs were significantly different between diagnosis and relapse. For B cell lymphoma, 8 microRNAs had differential expression in the non-remission group compared to dogs that completed CHOP in complete remission. Four of these microRNAs were also altered in patients that died prior to one-year. Kaplan-Meier survival curves for high versus low microRNA expression revealed that 10 microRNAs were correlated with progression-free survival and 3 with overall survival. This study highlights microRNAs of interest for canine multicentric lymphoma. Future goals include development of microRNA panels that may be useful as biomarkers with the intent to provide improved outcome prediction to veterinary cancer patients.

Osteosarcoma in the Post Genome Era: Preclinical Models and Approaches to Identify Tractable Therapeutic Targets.

PURPOSE OF REVIEW: Osteosarcoma (OS) is the most common cancer of bone, yet is classified as a rare cancer. Treatment and outcomes for OS have not substantively changed in several decades. While the decoding of the OS genome greatly advanced the understanding of the mutational landscape of OS, immediately actionable therapeutic targets were not apparent. Here we describe recent preclinical models that can be leveraged to identify, test, and prioritize therapeutic candidates. RECENT FINDINGS: The generation of multiple high fidelity murine models of OS, the spontaneous disease that arises in pet dogs, and the establishment of a diverse collection of patient-derived OS xenografts provide a robust preclinical platform for OS. These models enable evidence to be accumulated across multiple stages of preclinical evaluation. Chemical and genetic screening has identified therapeutic targets, often demonstrating cross species activity. Clinical trials in both PDX models and in canine OS have effectively tested new therapies for prioritization. Improving clinical outcomes in OS has proven elusive. The integrated target discovery and testing possible through a cross species platform provides validation of a putative target and may enable the rigorous evaluation of new therapies in models where endpoints can be rapidly assessed.

Flow Cytometric Detection of Circulating Osteosarcoma Cells in Dogs.

Osteosarcoma (OSA) is a malignant tumor of middle-aged dogs and adolescent humans. The clinical outcome of OSA has not improved over more than three decades, and dogs typically succumb to metastatic disease within 6 months despite tumor resection through limb amputation and adjuvant chemotherapy. Therefore, undetectable tumor cells with potential to form metastases are present at diagnosis. An assay to identify canine immortalized and primary OSA cells through flow cytometric detection of intracellular collagen 1 (Col I) and osteocalcin was optimized, and applied to blood samples from tumor-bearing dogs for detection of circulating tumor cells (CTCs). Spiking variable number of OSA cells into normal dog blood recovered 50-60% of Col I positive cells with high forward and variable side light scatter. An algorithm to exclude nonviable, doublet, and autofluorescent cells was applied to sequential blood samples from three dogs obtained prior to and after limb amputation, and at approximately, triweekly intervals over 121, 142, and 183 days of chemotherapy, respectively. Dogs had >100 CTC/106 leukocytes prior to amputation, variably frequent CTC during chemotherapy, and an increase up to 4,000 CTC/106 leukocytes within 4 weeks before overt metastases or death. Sorted CTCs were morphologically similar to direct tumor aspirates and positive for Col I. Although preliminary, findings suggest that CTCs are frequent in canine OSA, more numerous than carcinoma CTC in humans, and that an increase in CTC frequency may herald clinical deterioration. This assay may enable enumeration and isolation of OSA CTC for prognostic and functional studies, respectively. © 2019 International Society for Advancement of Cytometry.

Evaluation of effects of radiation therapy combined with either pamidronate or zoledronate on canine osteosarcoma cells.

Canine osteosarcoma is a devastating disease with an overall poor prognosis. Radiation therapy and bisphosphonates are currently used in combination for palliative treatment, despite a paucity of literature that investigates their combined use. The objectives of this study were to assess the in vitro effects of radiation therapy and bisphosphonates on canine osteosarcoma cells when used in combination. Canine osteosarcoma cell lines D17 and Dharma were treated with radiation and pamidronate or zoledronate, both alone and in combination. The effects of these treatments were assessed using clonogenic survival and cell viability assays. Dose-dependent decreases in clonogenic survival and cell viability were observed for both radiation and bisphosphonate treatment. Combination index analysis revealed antagonistic interactions when radiation and bisphosphonates were used in combination at specific doses for both D17 and Dharma osteosarcoma cells. Further investigation of the combined effects of radiation and bisphosphonates for the palliative treatment of canine osteosarcoma is warranted.

L'ostéosarcome canin est une maladie dévastatrice, avec un mauvais pronostic global. La radiothérapie et les bisphosphonates sont actuellement utilisés en combinaison pour le traitement palliatif, malgré une littérature limitée qui étudie leur utilisation combinée. Les objectifs de cette étude étaient d'évaluer les effets in vitro de la radiothérapie et des bisphosphonates sur des cellules d'ostéosarcome canin en combinaison. Les lignées cellulaires d'ostéosarcome canin D17 et Dharma ont été traitées par irradiation, le pamidronate et le zolédronate seuls et en association. Les effets de ces traitements ont été évalués en utilisant des tests de survie clonogénique et de viabilité cellulaire. Des diminutions dépendantes de la dose de la survie clonogénique et de la viabilité cellulaire ont été observées pour le traitement par rayonnement et par bisphosphonate. L'analyse de l'indice de combinaison a révélé des interactions antagonistes lorsque les radiations et les bisphosphonates étaient utilisés en association à des doses spécifiques, pour les cellules de D17 et d'ostéosarcome de Dharma. Une étude plus approfondie des effets combinés des rayonnements et des bisphosphonates pour le traitement palliatif de l'ostéosarcome canin est justifiée.(Traduit par les auteurs).