RNA aggregates harness the danger response for potent cancer immunotherapy.

Cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Here, we create "onion-like" multi-lamellar RNA lipid particle aggregates (LPAs) to substantially enhance the payload packaging and immunogenicity of tumor mRNA antigens. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for Toll-like receptor engagement in immune cells, systemically administered RNA-LPAs activate RIG-I in stromal cells, eliciting massive cytokine/chemokine response and dendritic cell/lymphocyte trafficking that provokes cancer immunogenicity and mediates rejection of both early- and late-stage murine tumor models. In client-owned canines with terminal gliomas, RNA-LPAs improved survivorship and reprogrammed the TME, which became "hot" within days of a single infusion. In a first-in-human trial, RNA-LPAs elicited rapid cytokine/chemokine release, immune activation/trafficking, tissue-confirmed pseudoprogression, and glioma-specific immune responses in glioblastoma patients. These data support RNA-LPAs as a new technology that simultaneously reprograms the TME while eliciting rapid and enduring cancer immunotherapy.

Safety of TCMCB07, a melanocortin-4 antagonist peptide, in dogs with naturally occurring cachexia.

BACKGROUND: The melanocortin 4 antagonist TCMCB07 is safe and effective in reversing cachexia caused by sepsis or cancer in rodents. The safety and pharmacokinetics of TCMCB07 are demonstrated in healthy beagle dogs. HYPOTHESIS/OBJECTIVES: The objectives of this study were to investigate the safety, peak plasma concentrations, and potential for efficacy of TCMCB07 in pet dogs with naturally occurring cachexia over a 4-week time period. ANIMALS: Fourteen dogs with cachexia of any underlying cause, except cancer of the oral cavity or gastrointestinal tract, were eligible for enrollment with informed client consent. METHODS: This study was a prospective, 1-armed open-label trial. Physical examination, complete blood count, chemistry panel, and owner-assessed quality of life surveys were checked at weeks 1, 2, and 4. Due to potential for bradycardia and hypotension, Holter monitoring and blood pressure evaluations were scheduled at pre-enrollment and week 4. RESULTS: Fourteen dogs completed the trial. Significant changes detected included increased mean body weight (18.6-19.5 kg, P < .02), increased body condition score (median Tufts 5-point thin dog scale score P < .004 and WSAVA muscle condition score P < .02) and increased mean blood urea nitrogen (21.79-30.43 mg dL-1 , P < .004). On quality of life surveys, pet owners perceived their dog appeared to be panting less (P < .002) and that the general health improved (P < .03). Four dogs had a change in coat pigmentation. The peak plasma concentration of TCMCB07 in cachectic dogs was similar to that in healthy beagle dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: TCMCB07 was safe and has potential efficacy in pet dogs with cachexia.

Characterization of expression and prognostic implications of GD2 and GD3 synthase in canine histiocytic sarcoma.

GD2 and GD3 are disialoganglioside oncofetal antigens important in oncogenesis. GD2 synthase (GD2S) and GD3 synthase (GD3S) are needed for GD2 and GD3 production. The objectives of this study are to validate the use of RNA in situ hybridization (RNAscope®) in the detection of GD2S and GD3S in canine histiocytic sarcoma (HS) in vitro and optimize this technique in canine formalin-fixed paraffin-embedded (FFPE) tissues. A secondary objective is to evaluate the prognostic significance of GD2S and GD3S on survival. Quantitative RT-PCR compared GD2S and GD3S mRNA expression between three HS cell lines followed by RNAscope® in fixed cell pellets from the DH82 cell line and FFPE tissues. Variables prognostic for survival were determined with Cox proportional hazard model. RNAscope® was validated for detection of GD2S and GD3S and optimized in FFPE tissues. GD2S and GD3S mRNA expression was variable between cell lines. GD2S and GD3S mRNA expression was detected and measured in all tumor tissues; there was no association with prognosis. GD2S and GD3S are expressed in canine HS and successfully detected using the high throughput technique of RNAscope® in FFPE samples. This study provides the foundation for future prospective research of GD2S and GD3S utilizing RNAscope®.

mRNA challenge predicts brain cancer immunogenicity and response to checkpoint inhibitors.

To prospectively determine whether brain tumors will respond to immune checkpoint inhibitors (ICIs), we developed a novel mRNA vaccine as a viral mimic to elucidate cytokine release from brain cancer cells in vitro. Our results indicate that cytokine signatures following mRNA challenge differ substantially from ICI responsive versus non-responsive murine tumors. These findings allow for creation of a diagnostic assay to quickly assess brain tumor immunogenicity, allowing for informed treatment with ICI or lack thereof in poorly immunogenic settings.

mRNA aggregates harness danger response for potent cancer immunotherapy.

Messenger RNA (mRNA) has emerged as a remarkable tool for COVID-19 prevention but its use for induction of therapeutic cancer immunotherapy remains limited by poor antigenicity and a regulatory tumor microenvironment (TME). Herein, we develop a facile approach for substantially enhancing immunogenicity of tumor-derived mRNA in lipid-particle (LP) delivery systems. By using mRNA as a molecular bridge with ultrapure liposomes and foregoing helper lipids, we promote the formation of 'onion-like' multi-lamellar RNA-LP aggregates (LPA). Intravenous administration of RNA-LPAs mimics infectious emboli and elicits massive DC/T cell mobilization into lymphoid tissues provoking cancer immunogenicity and mediating rejection of both early and late-stage murine tumor models. Unlike current mRNA vaccine designs that rely on payload packaging into nanoparticle cores for toll-like receptor engagement, RNA-LPAs stimulate intracellular pathogen recognition receptors (RIG-I) and reprogram the TME thus enabling therapeutic T cell activity. RNA-LPAs were safe in acute/chronic murine GLP toxicology studies and immunologically active in client-owned canines with terminal gliomas. In an early phase first-in-human trial for patients with glioblastoma, we show that RNA-LPAs encoding for tumor-associated antigens elicit rapid induction of pro-inflammatory cytokines, mobilization/activation of monocytes and lymphocytes, and expansion of antigen-specific T cell immunity. These data support the use of RNA-LPAs as novel tools to elicit and sustain immune responses against poorly immunogenic tumors.

Characterization of expression and prognostic implications of transforming growth factor beta, programmed death-ligand 1, and T regulatory cells in canine histiocytic sarcoma.

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-ß), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-ß, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope®). After validation was performed, RNAscope® on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO® software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-ß and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope® method was validated, TGF-ß H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-ß dots/cell. In summary, RNAscope® is a valid technique to detect TGF-ß and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-ß and PD-L1. Increased TGF-ß and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-ß, PD-L1, and Tregs effect on prognosis.

RNA in situ hybridisation as a molecular diagnostic technique targeting IBA-1 and CD204 in canine histiocytic sarcoma.

BACKGROUND: Canine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA-1, ionised calcium-binding adapter molecule 1, and CD204, a macrophage scavenger receptor, have been shown to be specific immunohistochemical markers helpful in distinguishing HS from other tumour types with similar morphological features. OBJECTIVES: This study was performed to demonstrate the use of RNA in situ hybridisation (ISH) technology allowing single-molecule RNA visualisation in formalin-fixed paraffin-embedded (FFPE) tissues as a molecular tool for the diagnosis of canine HS. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis for IBA-1 and CD204 were performed to correlate gene expression and protein expression of these two markers in the histiocytic sarcoma DH82 cell line. RNA-ISH for IBA-1 and CD204 was performed on the DH82 cell line to validate the RNA-ISH probes. RNA-ISH and immunohistochemistry (IHC) were performed in clinical HS FFPE samples to demonstrate mRNA and protein expression of IBA-1 and CD204. FFPE archived samples of canine round cell tumours, melanoma and anaplastic sarcoma were used as negative controls. RESULTS: RNA-ISH and IHC showed moderate to strong expression for IBA-1 and CD204 in the neoplastic cells in both the canine DH82 cell line and the archived canine HS samples. RNA-ISH and IHC showed scattered positive staining in the control tumours samples, consistent with macrophagic infiltration. CONCLUSION: RNA-ISH for CD204 and IBA-1 appeared to have a high specificity and sensitivity in our samples and may be an additional valuable diagnostic technique in identifying HS.

Identification of the interleukin-8 (CXCL-8) pathway in feline oral squamous cell carcinoma - A pilot study.

The purpose of this pilot study was to detect the presence of interleukin-8 (IL-8) and the potential downstream effects of IL-8 receptor activation in 2 previously characterized feline oral squamous cell carcinoma cell lines (SCCF1 and SCCF2). Interleukin-8 messenger RNA (mRNA) was initially detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). A previously validated and commercially available enzyme-linked immunosorbent assay (ELISA) test was used to measure IL-8 production in the supernatant of the 2 cell lines. Western blot was used to detect phosphorylation of proteins (AKT, ERK1/2, JAK2, STAT3, and Src), known to be downstream of interleukin-8 receptor activation. The IL-8 receptor-specific antagonists, Reparixin and SCH527123, were used to identify effects on phosphorylation of these proteins. Interleukin-8 mRNA and protein were detected in both SCCF1 and SCCF2 by RT-PCR and ELISA, respectively. Phosphorylation of ERK1/2, STAT3, and Src was detected in both cell lines. Inhibition of the IL-8 receptor led to a decrease in phosphorylation of Src, but not ERK1/2 or STAT3. In conclusion, feline squamous cell carcinoma cell lines can produce IL-8. Phosphorylation of Src seems, at least in part, a consequence of IL-8 receptor activation. The phosphorylation of ERK1/2 and STAT3, although present, seems independent of IL-8 receptor activation. Due to its potential effects on the tumor microenvironment, in addition to its autocrine effects on Src phosphorylation, the inhibition of the IL-8 receptor may become a beneficial therapeutic tool. Evaluation of the presence of both IL-8 and Src in many cases should elucidate their importance.

Le but de cette étude pilote était de détecter la présence d'interleukine-8 (IL-8) et les effets potentiels en aval de l'activation du récepteur IL-8 dans deux lignées cellulaires de carcinome épidermoïde oral félin (SCCF1 et SCCF2) précédemment caractérisées. L'ARN messager de l'interleukine-8 (ARNm) a été initialement détecté par amplification en chaîne par la polymérase à transcription inverse quantitative (qRT-PCR). Un test immuno-enzymatique ELISA précédemment validé et disponible dans le commerce a été utilisé pour mesurer la production d'IL-8 dans le surnageant des deux lignées cellulaires. L'immunobuvardage a été utilisé pour détecter la phosphorylation des protéines (AKT, ERK1/2, JAK2, STAT3 et Src), connues pour être en aval de l'activation du récepteur de l'interleukine-8. Les antagonistes spécifiques du récepteur IL-8, Reparixin et SCH527123, ont été utilisés pour identifier les effets sur la phosphorylation de ces protéines. L'ARNm et la protéine de l'interleukine-8 ont été détectés dans SCCF1 et SCCF2 par RT-PCR et ELISA, respectivement. La phosphorylation de ERK1/2, STAT3 et Src a été détectée dans les deux lignées cellulaires. L'inhibition du récepteur IL-8 a conduit à une diminution de la phosphorylation de Src, mais pas ERK1/2 ou STAT3. En conclusion, les lignées cellulaires de carcinome épidermoïde félin sont capables de produire de l'IL-8. La phosphorylation de Src semble, au moins en partie, une conséquence de l'activation du récepteur IL-8. La phosphorylation de ERK1/2 et STAT3, bien que présente, semble indépendante de l'activation du récepteur IL-8. En raison de ses effets potentiels sur le micro-environnement tumoral, en plus de ses effets autocrines sur la phosphorylation de Src, l'inhibition du récepteur IL-8 peut devenir un outil thérapeutique bénéfique. L'évaluation de la présence à la fois d'IL-8 et de Src dans un grand nombre de cas devrait élucider leur importance.(Traduit par Docteur Serge Messier).

Novel application of single-cell next-generation sequencing for determination of intratumoral heterogeneity of canine osteosarcoma cell lines.

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.

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

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.

GD2 chimeric antigen receptor modified T cells in synergy with sub-toxic level of doxorubicin targeting osteosarcomas.

Since the prognosis for children with high-risk osteosarcoma (OS) remains suboptimal despite intensive multi-modality therapies, there is a clear and urgent need for the development of targeted therapeutics against these refractory malignancies. Chimeric antigen receptor (CAR) modified T cells can meet this need by utilizing the immune system's potent cytotoxic mechanisms against tumor specific antigen targets with exquisite specificity. Since OS highly expresses the GD2 antigen, a viable immunotherapeutic target, we sought to assess if CAR modified T cells targeting GD2 could induce cytotoxicity against OS tumor cells. We demonstrated that the GD2 CAR modified T cells were highly efficacious for inducing OS tumor cell death. Interestingly, the OS cells were induced to up-regulate expression of PD-L1 upon interaction with GD2 CAR modified T cells, and the specific interaction induced CAR T cells to overexpress the exhaustion marker PD-1 along with increased CAR T cell apoptosis. To further potentiate CAR T cell killing activity against OS, we demonstrated that suboptimal chemotherapeutic treatment with doxorubicin can synergize with CAR T cells to effectively kill OS tumor cells.

Plasma ammonia concentration after L-asparaginase therapy in 27 dogs with high-grade lymphoma or leukemia.

OBJECTIVES: To establish the occurrence of increased plasma ammonia concentration after L-asparaginase (L-asp) administration in dogs with high-grade lymphoma or leukemia; to identify risk factors for the development of hyperammonemia after L-asp administration; and to determine occurrence of adverse events related to hyperammonemia. DESIGN: Prospective case controlled study of sequentially enrolled dogs between May 2011 and March 2012. SETTING: A university veterinary teaching hospital. ANIMALS: Twenty-seven dogs with high-grade lymphoma or leukemia. INTERVENTIONS: All dogs received L-asp intramuscularly at a median dose of 400 IU/kg. MEASUREMENTS AND MAIN RESULTS: Plasma ammonia concentrations were measured at baseline, 16 hours, and 48 hours after L-asp therapy. Clinicopathological abnormalities were assessed to determine risk factors for the development of hyperammonemia. Adverse events following L-asp were recorded. Median plasma ammonia concentrations at baseline, 16 hours, and 48 hours were 26 µmol/L (44 µg/dL), 98 µmol/L (166.9 µg/dL), and 67 µmol/L (114 µg/dL), respectively. Median plasma ammonia concentrations at 16 and 48 hours after administration were significantly increased compared to baseline. Six dogs had adverse events following L-asp administration. No significant clinical signs were noted that could clearly be attributed to hyperammonemia. No risk factors for developing hyperammonemia were identified; however, there was a positive correlation between the development of hyperammonemia at 16- and 48-hour time points. CONCLUSIONS: Subclinical hyperammonemia in dogs with lymphoma or leukemia after L-asp administration appears to be common. No risk factors were identified for the development of hyperammonemia after L-asp treatment, and severe adverse events were rare.

Retrospective Evaluation of Whole Body Computed Tomography for Tumor Staging in Dogs with Primary Appendicular Osteosarcoma.

OBJECTIVE: To evaluate whole body computed tomography (CT) for staging canine appendicular osteosarcoma. STUDY DESIGN: Retrospective case series. ANIMALS: Client-owned dogs diagnosed with appendicular osteosarcoma (n=39). METHODS: Medical records for client-owned dogs diagnosed with appendicular osteosarcoma from August 2008 to July 2014 were reviewed. Dogs were included if they had a confirmed diagnosis of appendicular osteosarcoma and were staged using whole body CT. Data collected included signalment, body weight, primary tumor location, serum alkaline phosphatase (ALP) activity, findings on 3-view thoracic radiographs, cytologic or histologic results, and findings on CT. RESULTS: Thirty-nine dogs (median age 8.5 years; median body weight 37 kg) had osteosarcoma of the distal radius (n=17), proximal humerus (11) and other sites. Serum ALP activity was elevated in 14 dogs. Bone metastasis was not detected in any dog on whole body CT. Pulmonary metastasis was considered definitive on CT based on board certified radiologist assessment in 2/39 dogs (5%). Two additional dogs (2/39, 5%) had soft tissue masses diagnosed on CT, consistent with concurrent, non-metastatic malignancies. CONCLUSION: Bone metastases were not identified in any dog with whole body CT. Thoracic and abdominal CT detected lung lesions and concurrent neoplasia in dogs with primary appendicular osteosarcoma. Whole body CT may be a useful adjunct to other screening tests for disseminated malignancy.

Clinical Response and Adverse Event Profile of Bleomycin Chemotherapy for Canine Multicentric Lymphoma.

Although canine multicentric lymphoma is initially responsive to multidrug chemotherapy, resistance and relapse create a need for novel chemotherapeutics. Bleomycin is an antitumor antibiotic with a minimal adverse event profile; though commonly used for human non-Hodgkin's lymphoma, its use is poorly characterized in dogs. The purpose of this retrospective case series was to describe the clinical response and adverse event profile of systemic bleomycin for canine multicentric lymphoma (n = 10). A partial response was noted in one dog that died 24 days later due to unrelated disease. Adverse events were infrequent and limited to grade 1 gastrointestinal and grade 1 constitutional toxicity. Although clinical response was minimal, systemic bleomycin was well tolerated when administered at 0.5 U/kg. Additional studies are warranted to determine the influence of administration schedule and dose on the efficacy of bleomycin for veterinary neoplasia.

ASSOCIATION BETWEEN COMPUTED TOMOGRAPHIC CHARACTERISTICS AND FRACTURES FOLLOWING STEREOTACTIC RADIOSURGERY IN DOGS WITH APPENDICULAR OSTEOSARCOMA.

The objective of this observational, descriptive, retrospective study was to report CT characteristics associated with fractures following stereotactic radiosurgery in canine patients with appendicular osteosarcoma. Medical records (1999 and 2012) of dogs that had a diagnosis of appendicular osteosarcoma and undergone stereotactic radiosurgery were reviewed. Dogs were included in the study if they had undergone stereotactic radiosurgery for an aggressive bone lesion with follow-up information regarding fracture status, toxicity, and date and cause of death. Computed tomography details, staging, chemotherapy, toxicity, fracture status and survival data were recorded. Overall median survival time (MST) and fracture rates of treated dogs were calculated. CT characteristics were evaluated for association with time to fracture. Forty-six dogs met inclusion criteria. The median overall survival time was 9.7 months (95% CI: 6.9-14.3 months). The fracture-free rates at 3, 6, and 9 months were 73%, 44%, and 38% (95% CI: 60-86%, 29-60%, and 22-54%), respectively. The region of bone affected was significantly associated with time to fracture. The median time to fracture was 4.2 months in dogs with subchondral bone involvement and 16.3 months in dogs without subchondral bone involvement (P-value = 0.027, log-rank test). Acute and late skin effects were present in 58% and 16% of patients, respectively. Findings demonstrated a need for improved patient selection for this procedure, which can be aided by CT-based prognostic factors to predict the likelihood of fracture.

OUTCOMES AND PROGNOSTIC FACTORS ASSOCIATED WITH CANINE SINONASAL TUMORS TREATED WITH CURATIVE INTENT CONE-BASED STEREOTACTIC RADIOSURGERY (1999-2013).

Stereotactic radiosurgery (SRS) is a relatively new therapeutic option in veterinary oncology. The role of this modality has not been extensively evaluated for the use in canine nasal tumors. The objective of this retrospective, observational study was to describe the clinical outcome and prognostic factors associated with survival times in a sample of canine patients treated with SRS for sinonasal tumors. Fifty-seven dogs with sinonasal tumors met inclusion criteria. Histologic diagnoses included sarcoma (SA) (n = 9), carcinoma (CA) (n = 40), osteosarcoma (OSA) (n = 7), and round cell (n = 1). Four of 57 cases were treated twice with SRS. For these, the median and mean doses delivered were 30Gy and 33Gy, respectively (range 18.75Gy-56Gy). Late effects occurred in 23 cases and ranged from grades I-III. The median overall survival time was 8.5 months. The median overall survival times in dogs with tumor type of CA, SA, and OSA were 10.4, 10.7, and 3.1 months, respectively. Dogs with the tumor type of OSA had shorter overall survival time than that in dogs with tumor type of CA and SA. Findings from this retrospective study indicated that SRS may be beneficial for canine patients with sinonasal tumors, however a controlled clinical trial would be needed to confirm this. Prospective studies are also needed to better define the role of SRS as palliative or curative, and to further investigate the risk of clinically significant toxicity.

Synthesis of polymeric phosphonates for selective delivery of radionuclides to osteosarcoma.

Discussed in detail is the synthesis and primary structure characterization of two polymers aimed at advancing the treatment of pediatric osteosarcoma. These polymers are designed to systemically deliver radiometals specifically to osteosarcomas using the passive targeting mechanism of enhanced permeability and retention (the EPR effect). The approach begins with the synthesis of a polymer capable of binding radiometals, for which prior data show improved site-specific targeting of solid tumors. Building on this success, a second polymer has been designed for improving the efficacy of currently available radionuclide therapies by incorporating the FDA-approved small-molecule ligand Quadramet directly onto the polymer structure. Time-activity curves of the phosphonate-functionalized polymers show rapid clearance from the central compartment and nontargeted organs, with up to 65% of injected activity being excreted within 3 hours. Both polymer ligands demonstrate good osteosarcoma targeting capability with little to no uptake in organs associated with the dose-limiting bone marrow. Additionally, biodistribution studies in nonosseous tumor models demonstrate the tumor targeting mechanism of the polymer ligands, which appears to be influenced by the high affinity of the phosphonate functionality for the positively charged hydroxyapatite mineral found in bone tumors.

Stereotactic radiosurgery and fracture fixation in 6 dogs with appendicular osteosarcoma.

OBJECTIVE: To evaluate clinical outcome of dogs with appendicular osteosarcoma (OSA) treated with stereotactic radiosurgery (SRS) and subsequent internal fixation of a pathologic fracture. STUDY DESIGN: Retrospective case series. ANIMALS: Dogs with spontaneous-occurring appendicular OSA (n = 6). METHODS: Medical records (May 2002-January 2008) of dogs that had SRS for appendicular OSA were reviewed. Dogs were included if they had a pathologic fracture either before or after SRS and were treated with internal fixation. Signalment, history, physical examination findings, clinicopathologic data, diagnostic imaging findings, biopsy results, surgical complications, number of surgeries, adjuvant therapy, development of metastatic disease and cause of death were recorded. RESULTS: Six dogs met the inclusion criteria. Two dogs had a pathologic fracture at admission and 4 dogs developed a fracture after SRS with a mean ± SD time to fracture development of 6.25 ± 1.65 months. The first 3 fractures were repaired using an open approach and the latter three using minimally invasive percutaneous osteosynthesis (MIPO). Infection occurred in 5 dogs and implant failure in 3. Limb function was subjectively assessed as good in all dogs when the implants were stable and infections were subclinical. Survival times ranged from 364-897 days; 1 dog was lost to follow-up. CONCLUSIONS: Fracture repair using internal fixation should be considered a viable limb-sparing alternative for pathologic fractures that have been treated with SRS.

Effects of aurothiomalate treatment on canine osteosarcoma in a murine xenograft model.

Osteosarcoma is a highly fatal cancer, with most patients ultimately succumbing to metastatic disease. The purpose of this study was to evaluate the effects of the antirheumatoid drug aurothiomalate on canine and human osteosarcoma cells and on canine osteosarcoma growth and metastasis in a mouse xenograft model. We hypothesized that aurothiomalate would decrease osteosarcoma cell survival, tumor cellular proliferation, tumor growth, and metastasis. After performing clonogenic assays, aurothiomalate or a placebo was administered to 54 mice inoculated with canine osteosarcoma. Survival, tumor growth, embolization, metastasis, histopathology, cell proliferation marker Ki67, and apoptosis marker caspase-3 were compared between groups. Statistical analysis was carried out using the Kaplan-Meier method with the log-rank test and one-way analysis of variance with the Tukey's test or Dunn's method. Aurothiomalate caused dose-dependent inhibition of osteosarcoma cell survival (P<0.001) and decreased tumor growth (P<0.001). Pulmonary macrometastasis and Ki67 labeling were reduced with low-dose aurothiomalate (P=0.033 and 0.005, respectively), and tumor emboli and pulmonary micrometastases were decreased with high-dose aurothiomalate (P=0.010 and 0.011, respectively). There was no difference in survival, tumor development, ulceration, mitotic indices, tumor necrosis, nonpulmonary metastases, and caspase-3 labeling. Aurothiomalate treatment inhibited osteosarcoma cell survival and reduced tumor cell proliferation, growth, embolization, and pulmonary metastasis. Given aurothiomalate's established utility in canine and human medicine, our results suggest that this compound may hold promise as an adjunctive therapy for osteosarcoma. Further translational research is warranted to better characterize the dose response of canine and human osteosarcoma to aurothiomalate.

Partial cytogenetic response with toceranib and prednisone treatment in a young dog with chronic monocytic leukemia.

Treatment of chronic monocytic leukemia (CMoL) in dogs has traditionally consisted of hydroxyurea. The use of tyrosine kinase inhibitors has been proposed as a treatment option for dogs with CMoL but has never been reported. We report a case of CMoL in a young dog that achieved clinical remission with treatment with the tyrosine kinase inhibitor toceranib and prednisone.