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Diagnosis of gastric tumors in dogs is difficult and is often obtained by biopsy following identification of a mass through ultrasound (US) or endoscopy. In human medicine, modalities such as CT and endoscopy are standard of care in the diagnosis and staging of gastric tumors. Although one veterinary study has described CT findings of gastric tumors in dogs using iatrogenic gas dilation, there are no veterinary studies that have directly compared the usefulness of US versus CT in the diagnosis and staging of these tumors. This retrospective, descriptive study evaluated US and CT images from 13 dogs. Gastric tumor diagnoses included leiomyoma (n = 4), adenocarcinoma (n = 3), leiomyosarcoma (n = 3), gastrointestinal stromal tumor (n = 2), and lymphoma (n = 1). Computed tomography was successful in identification of 92% of gastric tumors, while US identified only 69%. Computed tomography identified more locations of lymphadenopathy and correctly identified the location of gastric tumors more frequently than US when compared to the surgical, endoscopic, or necropsy reports. Most features seen on US and CT overlapped between the different tumor types. Lymphoma had a lower mean attenuation in CT than the other gastric tumors and was the only gastric tumor to not have complete loss of the gastric wall layering on US. As expected, adenocarcinoma appeared as gastric wall thickening with regional lymphadenopathy. Findings supported using CT as an ancillary diagnostic test for characterizing and staging gastric tumors in dogs and assisting in the selection of surgical candidates.

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.

Carboplatin is a platinum chemotherapeutic agent commonly used in veterinary oncology that is currently classified as an irritant to local tissues when extravasated. To the authors’ knowledge, there are no reports of vesicant injuries associated with carboplatin administration reported in the veterinary literature. In this case series, seven dogs are described to have experienced injuries following a suspected carboplatin extravasation resembling vesicant injuries a median of 7 days after carboplatin administration (range 4–15 days). Wounds healed with a variety of treatments, including medical management and/or surgical debridement, a median of 25.5 days (range 7–49 days) after observation of the suspected extravasation injury. There were no obvious similarities involving carboplatin administration among patients to explain why these reactions occurred. Extravasation injury should be considered a possible local complication associated with carboplatin chemotherapy.

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.

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.

Stereotactic radiation therapy (SRT) has emerged as a convenient definitive treatment modality in veterinary medicine, but few studies exist evaluating outcome with treatment for canine nasal tumors, and no studies report the treatment of one single tumor histotype. This retrospective, observational study evaluates toxicity, response, and survival in 17 dogs with nasal carcinomas treated with SRT. Dogs received a median of 3000 centigray in three fractions via 6-MV linear accelerator. Eighty-eight percent of patients (n = 15) demonstrated clinical benefit. Of dogs with repeated CT imaging (n = 10), 60% (n = 6) achieved a partial response and 10% (n = 1) achieved a complete response. Median progression-free survival (PFS) was 359 days. Median survival time (MST) was 563 days. Among dogs evaluable for acute toxicity, 50% (n = 10) developed low grade toxicity (grade 1, n = 4; grade 2, n = 1). No patients developed grade 3 toxicity. 16 dogs (87%) evaluable over the long term developed signs consistent with possible late toxicity. The majority of late toxicities were mild (alopecia, hyperpigmentation, and leukotrichia n = 10; ocular discharge and keratoconjunctivitis sicca n = 5). Thirty-seven percent of patients (n = 6) developed seven possible grade 3 late toxicities (blindness, n = 3; fistula, n = 1; seizures, n = 3), which were difficult to distinguish from progressive disease in most patients. Of the prognostic factors evaluated (demographics, tumor stage, dosimetric data, epistaxis, facial deformity, clinical response, image-based response, nonsteroidal anti-inflammatory drugs, and chemotherapy), only clinical response was a positive prognostic factor on MST (P < .00). No factors were found to be significantly associated with PFS.

Ponatinib is a broad-spectrum tyrosine kinase inhibitor that targets numerous receptor tyrosine kinases (RTKs), including but not limited to fibroblast growth factor receptor (FGFR)-1, platelet derived growth factor receptor (PDGFR)-α, and vascular endothelial growth factor receptor (VEGFR)-2. This study evaluated the expression of FGFR-1, PDGFR-α, and VEGFR-2 in three canine mast cell tumor (MCT) cell lines (CM-MC1, VI-MC1, CoMS) and the effects of ponatinib on these MCT cell lines. Quantitative RT-PCR confirmed the expression of FGFR-1, PDGFR-α, and VEGFR-2 in the three MCT cell lines. Ponatinib exhibited dose- and time-dependent cytotoxicity in MCT cell lines via MTT assay. The IC50 for 24, 48, and 72 h across the three cell lines ranged from 38.47 nM to 103.3 nM, which is clinically comparable to dose ranges established for humans. Significantly increased apoptosis in each cell line was seen between 12 and 18 h after treatment with IC50 of ponatinib via Annexin-V and Caspase-3/7 assays. These data suggest that ponatinib could be a possible therapeutic agent for canine MCTs. Further studies are needed to investigate the prognostic value of FGFR-1, PDGFR-α, and VEGFR-2 in canine MCTs.

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.

Fluorescence-activated cell sorting (FACS) is a branch of flow cytometry that allows for the isolation of specific cell populations that can then be further analyzed by single-cell RNA sequencing (scRNA-seq). When utilizing FACS for population isolation prior to sequencing, it is essential to consider the protection of RNA from RNase activity, environmental conditions, and the sorting efficiency to ensure optimum sample quality. This study aimed to optimize a previously published MDSC flow cytometry strategy to FACS sort canine Myeloid-Derived Suppressor Cells (MDSC) with various permutations of RNAlater ™ and RiboLock™ before and after FACS sorting. Concentrations of RNAlater™ greater than 2 % applied before flow analysis affected cell survival and fluorescence, whereas concentrations ≤ 2 % and time ≤ 4 h had little to no effect on cells. To shorten the procedural time and to enhance the sorting of rare populations, we used a primary PE-conjugated CD11b antibody and magnetic column. The combination of RiboLock™ pre- and post-sorting for FACS provided the best quality RNA as determined by the RNA integrity number (RIN ≥ 7) for scRNA-seq in a normal and dog and a dog with untreated oral melanoma dog. As proof of principle, we sequenced two samples, one from a normal dog another from a dog with untreated oral melanoma. Applying scRNA-Seq analysis using the 10X Genomic platform, we identified 6 clusters in the Seurat paired analysis of MDSC sorted samples. Two clusters, with the majority of the cells coming from the melanoma sample, had genes that were upregulated (> log2); these included MMP9, MMP1, HPGD, CPA3, and GATA3 and CYBB, CSTB, COX2, ATP6, and COX 17 for cluster 5 and 6 respectively. All genes have known associations with MDSCs. Further characterization using pathway analysis tools was not attempted due to the lower number of cells sequenced in the normal sample. The benefit deriving from the results of the study helped to gain data consistency when working with cells prone to RNase activity, and the scRNA-seq provided data showing transcriptional heterogeneity in MDSC populations and potentially identifying previously unreported or rare cell populations.

Vincristine is included in vincristine, cyclophosphamide, doxorubicin and prednisone (CHOP) chemotherapy protocols, which are the gold-standard treatment for high-grade canine lymphoma. Vincristine can result in relatively high rates of gastrointestinal toxicity, whereas vinblastine is generally well tolerated and thus may represent an under-utilized and minimally toxic alternative to vincristine. Our objective was to determine the response rate and toxicity associated with a single dose of vinblastine administered to dogs with treatment-naive, intermediate to large-cell, multicentric lymphoma. Twenty client-owned dogs were enrolled with signed owner consent. A Simon's minimax, phase II, two-stage trial was performed to test the efficacy of vinblastine administered at 2 mg/m2 IV followed by a pilot trial of vinblastine at 2.5 mg/m2 . No dogs were administered concurrent steroids or other chemotherapy. One out of 14 dogs receiving vinblastine at 2 mg/m2 demonstrated a partial response. Three out of five dogs demonstrated a partial response to vinblastine at 2.5 mg/m2 . Gastrointestinal toxicity was infrequent and low grade for both groups. The majority of dogs (80%) in the 2.5 mg/m2 dosing group developed neutropenia 1-week post administration. Vinblastine was well tolerated but minimally efficacious at a dose of 2 mg/m2 IV in dogs with treatment-naive, multicentric lymphoma. Because of poor response rates, treatment at this dose is not recommended. A small subset of dogs administered 2.5 mg/m2 had significantly improved response rates (P = 0.04), suggesting that higher doses may have improved efficacy, although further research is indicated to confirm these preliminary findings.

Melanoma in humans and canines is an aggressive and highly metastatic cancer. The mucosal forms in both species share genetic and histopathologic features, making dogs a valuable spontaneous disease animal model. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells of myeloid origin with immunosuppressive capabilities, which are increased in many human cancers and contribute to tumor immune evasion. They are a possible target to improve immunotherapy outcomes. Current information regarding MDSCs in canines is minimal, limiting their use as translational model for the study of MDSCs. The objective of this study was to characterize major MDSCs subsets (monocytic and polymorphonuclear) and the cytokines granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 10 (IL-10) and monocyte chemoattractant protein-1 (MCP-1) in canines with malignant melanoma and to evaluate changes in MDSCs and the cytokines over time in response to a GD3-based active immunotherapy. Whole blood and serum collected from 30 healthy controls and 33 patients enrolled in the University of Florida melanoma vaccine trial were analyzed by flow cytometry with canine specific CD11b, MHCII and anti-human CD14 antibodies to assess ostensibly polymorphonuclear-MDSC (CD11b+ MHCII- CD14-) and monocytic-MDSC (CD11b+ MHCII- CD14+) subsets. IL-10, MCP-1 and both MDSCs subsets were significantly elevated in melanoma dogs versus controls. Both MDSCs subsets decreased significantly following GD3-based immunotherapy administration but no significant changes in cytokines were seen over time. To our knowledge, this is the first report documenting increased monocytic-MDSCs in canine melanoma. This is consistent with human malignant melanoma data, supporting dogs as a valuable model for therapeutic intervention studies.

This retrospective case series evaluates the outcome of 21 dogs with grade II stage 2 mast cell tumour (MCT) treated with adequate local therapy and adjuvant systemic chemotherapy (prednisone, vinblastine and CCNU). The median survival for all dogs was 1359 days (range, 188-2340). Median disease-free interval was 2120 days (149-2325 days). Dogs treated with surgery and chemotherapy had shorter survival (median, 1103 days; 188-2010 days) than those that underwent surgery, radiation therapy and chemotherapy as part of their treatment (median, 2056 days; 300-2340 days). Two patients had local recurrence in the radiation field and four patients had de novo MCT. Distant metastasis was not observed in any dogs. The results of this study suggest that, in the presence of loco-regional lymph node metastasis in grade II MCT, the use of prednisone, vinblastine and CCNU after adequate local-regional therapy can provide a median survival in excess of 40 months.

Introduction: The disialyl gangliosides GD2/GD3 have been implicated in the enhancement of malignancy in a number of human and animal cancers and as a tumor antigen target for immunotherapy. In a recent abstract presented at an AACR Conference we reported on the coexpression of GD2/GD3 in four canine osteosarcoma (OSA) cell lines (1). In a prospective IACUC approved clinical trial we vaccinated dogs with a GD3-based vaccine with naturally occurring OSA receiving surgery and carboplatin chemotherapy to investigate the expression profiles of immune modulating cells overtime. Methods: Dogs will be entered into the study only if they meet the following inclusion criteria: have a confirmed diagnosis of OSA and no other life-threatening diseases. The study will accrue 40 cases; 20 will receive the vaccine plus standard of care and 20 dogs will receive only the standard of care (amputation and intent to treat with 6 doses of carboplatin). On admission blood will be collected according to the protocol for monitoring of the immune response. The dogs will then be vaccinated according to a predetermined protocol during chemotherapy and staged. The immune response profile of the vaccine group will be compared to dogs receiving standard of care alone and normal dogs. Flow cytometric platforms were developed to monitor changes in immune cells (CD5, CD21, CD4, CD8, CD14, CD11b, MHCII, and Foxp3). In addition, IHC arrays and RNA Scope will be developed for checkpoints of immunity PD1, PDL-1 and expression of intratumoral immune cells. Results: Currently twenty dogs with osteosarcoma have enrolled into the study and have received standard of care and vaccination. Complete flow cytometric immune profiles are available for nine dogs with osteosarcoma. On admission, all dogs with OSA showed elevated cell counts of Treg (FoxP3+/CD4+) cells, Monocytic (m-) and Granulocytic (g-) myeloid derived suppressor cells (MDSCs) when compared to normal dogs (all dogs had normal CBCs). All m-MDCSs and g-MDSCs and Treg cells decreased significantly after the first dose of chemotherapy. Serial sampling over weeks showed sustained inhibition even after chemotherapy was completed (18 weeks). Two OSA cases which relapsed with metastasis to the lungs showed significant increases in Treg cells at the time of restaging. Complete necropsy post-therapy in three dogs showed changes in metastatic profiles; 2/3 showed no metastatic disease to the lungs, but metastases occurred to bone and kidneys. Discussion: MDSCs series of cells and Treg cells are increased in OSA compared to normal dogs and may be a factor in maintaining a welcoming tumor microenvironment and resistance to immunotherapy. MDSCs are reported to be elevated in other cancers, but not in naturally occurring OSA. Furthermore, canine OSA cell lines show increased expression of CCL2 and COX2 (data not shown). Recruitment of MDSCs to the cancer micro-tumor environment are thought to be mediated by the chemokine CCL2 and COX2. Abrogation of these pathways with chemotherapy and possibly immunotherapy may enhance overall survival. Early necropsy data seem to support the attenuation of the metastatic profile in dogs receiving standard of care and GD3-based vaccine. Conclusions: Anticipated results from the study will be used to adjust the vaccine protocol according to the changes in immune profiles. Data is not mature enough to evaluate survival at this time. Acknowledgment: The study is funded by a grant from the American Kennel Club Health Foundation and The UF CVM. Reference: 1. Milner RJ, Chimura N, Bowles KD, Salute M. Abstract A29: Differential expression of the gangliosides GD3 and GD2 in canine and human osteosarcoma cell lines: An immunotherapy target. Cancer Immunol Res 2015 Oct 1;3(10 Supplement):A29. Citation Format: Bikash Sahay, Shana Hutchison, Matt Cascio, Amandine Lejeune, Carlos Souza, Anna Szivek, Keijiro Shiomitsu, Kayla Harding, Stacey Fox-Alvarez, Mia Livaccarri, Lindsay Powers, Rowan James Milner. Changes in immune profiles of osteosarcoma dogs receiving a GD3-based vaccine concurrently with carboplatin chemotherapy and surgery [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A07.

To evaluate whole body computed tomography (CT) for staging canine appendicular osteosarcoma.Retrospective case series.Client-owned dogs diagnosed with appendicular osteosarcoma (n=39).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.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.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.