Metagenomic analysis of DNA viruses with targeted sequence capture of canine lobular orbital adenomas and normal conjunctiva

Our study aims are: (1) to evaluate phenotypically normal canine conjunctival and orbital tissue and tissue from canine lobular orbital adenomas (CLOAs) for the presence of viral genomic material and (2) phylogenetically classify detected DNA viruses to determine if a DNA virus is associated with CLOAs. A total of 31 formalin fixed paraffin embedded CLOA tissue samples, 4 papillomas or sarcoid, and 10 fresh clinically normal conjunctival tissues were included in this study. Genomic DNA was isolated from all samples and sequencing libraries were prepared. The libraries were molecularly indexed and pooled and viral DNA was enriched via targeted sequence capture utilizing ViroCap. The libraries were sequenced on the Illumina HiSeq platform and compared to known viral DNA reference genomes to identify viral DNA. Carnivore parvovirus was identified in 6.4% and 20% of CLOA tissue and normal conjunctival samples, respectively. This study showed that conjunctival tissue from healthy dogs and CLOAs uncommonly harbor DNA viruses, and no DNA virus was associated with these tumors. Further studies are needed to evaluate the etiologic cause of CLOAs

6-Thioguanine and zebularine globally demethylate a canine lyphoma cell line

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Epigenetic modifications of chromatin, including methylation, histone modifications, and protein chaperoning, play a significant role in the biology of cancer. In concert with mutations, epigenetic modifications alter gene expression and protein translation/activity to confer a neoplastic phenotype. Hypermethylation of tumor suppressor genes or hypomethylation of proto-oncogenes or oncogenes can abrogate cell-cycle controls. These processes are reversible, resulting in restoration of normal gene expression. Relationships between methylation aberrations in human NHL have already been made; work is yet to be done for canine lymphoma. Recent treatment alterations are now being directed at specific epigenetic changes. Many of the common epigenetic modifiers have previously been used in both humans and dogs. This lends drugs already proven to be safe available for research opportunities in vivo. The purpose of this research project is to determine if epigenetically-active drugs could result in phenotypic reversion in a canine lymphoma cell line. To test this, we evaluated a cells in vitro, exposing them to demethylating agents. Our end analysis was generation of cell proliferation and viability assays, quantitation of global methylation levels, and confirmation of demethylating drug targets. We hypothesized that canine lymphoma cells will undergo global demethylation in vitro after treatment with demethylating agents including 6-thioguanine (6-TG) and zebularine (Zeb). As in humans, 6-TG will downregulate DNMT- 1. The demethylating agents will have a dose dependent effect on global methylation and cell kill. The canine lymphoma cell line, CLGL-90, was grown in vitro with custom RPMI media using sterile techniques. The cells were treated with control (buffer solution), 6-TG, or Zeb in varying concentrations. Initial cell counts were collected prior to the drugs being added, and at 24 and 48-hour time points. Half of the originally plated cells were harvested for methylation analysis and cell survival analysis at 24 hours and replaced with appropriate media. The remaining cells were then re-treated and removed for analysis at 48 hours. Methylation analysis was HpaI/MspII (selective and non-selective methylation restriction enzymes) ratio. Western blot was used to determine DNMT1 protein levels before and after treatment. Cell survival curves were calculated using hemocytometer and trypan blue staining. The CLGL-90 cells were globally demethylated with increasing concentrations of 6-TG and Zeb. DNMT1 was reduced in cells treated with 6-TG and Zeb compared to controls, in a dose-dependent manner. Increasing concentrations of the demethylating agents also resulted in decreased cell survival, with 6-TG more effective than Zeb. 6-TG and Zeb globally demethylate the CLGL-90 canine lymphoma cell line and result in decreased amounts of DNMT-1 protein present. Increasing concentrations of the drugs also results in increasing cell kill. Future directions include in vitro work using these drugs in concert with cytotoxic chemotherapy drugs against resistant lymphoma cells. We also plan to begin using these agents in vivo, targeting resistant canine lymphoma patients in the clinic

Long-Term Survival and Glycemic Control with Toceranib Phosphate and Prednisone for a Metastatic Canine Insulinoma

Canine insulinoma is a highly metastatic neoplasm that is associated with a guarded to poor prognosis in dogs with distant metastases. A median survival of 6 mo has been reported for dogs with metastatic insulinoma. The dog in this report, diagnosed with stage III pancreatic insulinoma, had long-term glycemic control with survival of over 24 mo while receiving prednisone and toceranib phosphate after partial pancreatectomy. Toceranib phosphate has been shown to be an efficacious therapy for canine mast cell tumors with increasing evidence that it may be beneficial in the medical management of neuroendocrine tumors

Fracture rate and time to fracture in dogs with appendicular osteosarcoma receiving finely fractionated compared to coarsely fractionated radiation therapy: A single institution study

Abstract Background Radiation therapy (RT) is used for local pain alleviation in dogs with appendicular osteosarcoma (OS), especially among dogs that are poor surgical candidates for amputation. However, many historical reports of fractionated protocols lack time to fracture and fracture rates. Objectives The primary objectives of this retrospective study were to determine fracture rate and time to fracture of dogs receiving RT (coarse or fine fractionated) for appendicular OS. Secondary objectives were to evaluate tolerability and disease outcome measures. Methods Fifty‐one dogs that received RT as part of treatment for appendicular OS were available for evaluation. Forty‐five received coarse fractionation (C‐RT, 8 or 6 Gy per fraction protocols [C‐RT8 or C‐RT6]) while the remaining six received fine fractionation (F‐RT). Results The overall pathologic fracture rate was 37%. Pathologic fracture rate was significantly higher for dogs that received F‐RT (5/6, 83%) compared to dogs that received C‐RT (12/40, 30%, p = 0.021). In the 17 dogs that fractured, the overall median time to fracture was 57 days. For all dogs, the median progression free interval (PFI) and median overall survival time (OST) were 90 and 140 days, respectively. In a very small cohort of dogs (n = 7) treated with zoledronate and RT, fracture rate was 0% and extended survival times were noted. Conclusions In conclusion, C‐RT is recommended over F‐RT due to lower risk of pathologic fracture and similar PFI. Prospective evaluation of combined C‐RT and zoledronate, especially for dogs with poor surgical candidacy, is warranted for the treatment of canine appendicular osteosarcoma

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

PurposeThe 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 methodsA 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.ResultsThere 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.ConclusionsIn 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

Clinical validation of a next-generation sequencing-based multi-cancer early detection "liquid biopsy" blood test in over 1,000 dogs using an independent testing set: The CANcer Detection in Dogs (CANDiD) study.

Cancer is the leading cause of death in dogs, yet there are no established screening paradigms for early detection. Liquid biopsy methods that interrogate cancer-derived genomic alterations in cell-free DNA in blood are being adopted for multi-cancer early detection in human medicine and are now available for veterinary use. The CANcer Detection in Dogs (CANDiD) study is an international, multi-center clinical study designed to validate the performance of a novel multi-cancer early detection "liquid biopsy" test developed for noninvasive detection and characterization of cancer in dogs using next-generation sequencing (NGS) of blood-derived DNA; study results are reported here. In total, 1,358 cancer-diagnosed and presumably cancer-free dogs were enrolled in the study, representing the range of breeds, weights, ages, and cancer types seen in routine clinical practice; 1,100 subjects met inclusion criteria for analysis and were used in the validation of the test. Overall, the liquid biopsy test demonstrated a 54.7% (95% CI: 49.3-60.0%) sensitivity and a 98.5% (95% CI: 97.0-99.3%) specificity. For three of the most aggressive canine cancers (lymphoma, hemangiosarcoma, osteosarcoma), the detection rate was 85.4% (95% CI: 78.4-90.9%); and for eight of the most common canine cancers (lymphoma, hemangiosarcoma, osteosarcoma, soft tissue sarcoma, mast cell tumor, mammary gland carcinoma, anal sac adenocarcinoma, malignant melanoma), the detection rate was 61.9% (95% CI: 55.3-68.1%). The test detected cancer signal in patients representing 30 distinct cancer types and provided a Cancer Signal Origin prediction for a subset of patients with hematological malignancies. Furthermore, the test accurately detected cancer signal in four presumably cancer-free subjects before the onset of clinical signs, further supporting the utility of liquid biopsy as an early detection test. Taken together, these findings demonstrate that NGS-based liquid biopsy can offer a novel option for noninvasive multi-cancer detection in dogs