Effect of proteasome inhibitors on canine lymphoma cell response to CHOP chemotherapy in vitro.

The standard treatment for canine lymphoma is the CHOP chemotherapy regimen. Proteasome inhibitors have been employed with CHOP for the treatment of human haematological malignancies but remain to be fully explored in canine lymphoma. We identified an association between poor response to CHOP chemotherapy and high mRNA expression levels of proteasomal subunits in a cohort of 15 canine lymphoma patients, and sought to determine the effect of proteasome inhibitors on the viability of a canine B-cell lymphoma cell line (CLBL-1). The aim of this study was to investigate whether proteasome inhibitors sensitize these cells to the CHOP agents doxorubicin, vincristine and cyclophosphamide (as 4-hydroxycyclophosphamide/4-HC). CLBL-1 cells were sensitive to proteasome inhibition by bortezomib and ixazomib. The IC50 of bortezomib was 15.1 nM and of ixazomib was 59.14 nM. Proteasome inhibitors plus doxorubicin had a synergistic effect on CLBL-1 viability; proteosome inhibitors plus vincristine showed different effects depending on the combination ratio, and there was an antagonistic effect with 4-HC. These results may have clinical utility, as proteasome inhibition could potentially be used with a synergizing CHOP compound to improve responsiveness to chemotherapy for canine lymphoma patients.

Renal tubule-specific Atgl deletion links kidney lipid metabolism to glucagon-like peptide 1 and insulin secretion independent of renal inflammation or lipotoxicity.

OBJECTIVE: Lipotoxic injury from renal lipid accumulation in obesity and type 2 diabetes (T2D) is implicated in associated kidney damage. However, models examining effects of renal ectopic lipid accumulation independent of obesity or T2D are lacking. We generated renal tubule-specific adipose triglyceride lipase knockout (RT-SAKO) mice to determine if this targeted triacylglycerol (TAG) over-storage affects glycemic control and kidney health. METHODS: Male and female RT-SAKO mice and their control littermates were tested for changes in glycemic control at 10-12 and 16-18 weeks of age. Markers of kidney health and blood lipid and hormone concentrations were analyzed. Kidney and blood lysophosphatidic acid (LPA) levels were measured, and a role for LPA in mediating impaired glycemic control was evaluated using the LPA receptor 1/3 inhibitor Ki-16425. RESULTS: All groups remained insulin sensitive, but 16- to 18-week-old male RT-SAKO mice became glucose intolerant, without developing kidney inflammation or fibrosis. Rather, these mice displayed lower circulating insulin and glucagon-like peptide 1 (GLP-1) levels. Impaired first-phase glucose-stimulated insulin secretion was detected and restored by Exendin-4. Kidney and blood LPA levels were elevated in older male but not female RT-SAKO mice, associated with increased kidney diacylglycerol kinase epsilon. Inhibition of LPA-mediated signaling restored serum GLP-1 levels, first-phase insulin secretion, and glucose tolerance. CONCLUSIONS: TAG over-storage alone is insufficient to cause renal tubule lipotoxicity. This work is the first to show that endogenously derived LPA modulates GLP-1 levels in vivo, demonstrating a new mechanism of kidney-gut-pancreas crosstalk to regulate insulin secretion and glucose homeostasis.

Kinetic analysis of oncolytic OrfV-induced innate and adaptive immune responses in a murine model of late-stage ovarian cancer.

Immunotherapies revive host immune responses against tumors by stimulating innate and adaptive immune effector cells with antitumor functions. Thus, detailed studies of immunological cell phenotypes and functions within the tumor microenvironment (TME) following immunotherapy treatments is critical to identifying the determinants of therapeutic success, optimizing treatment regimens, and driving curative outcomes. Oncolytic viruses such as Orf virus (OrfV) are multifunctional biologics that preferentially infect and kill cancer cells while simultaneously causing inflammation that drives anticancer immune responses. Here, we describe the immunological impact of OrfV on the ascites TME in a preclinical model of advanced-stage epithelial ovarian cancer. OrfV promoted the infiltration of several immune effector cells with increased expression of activation markers and effector cytokines into the ascites TME, which correlated with reduced ascites tumor burden and improved survival. The kinetics of the immune response and change in tumor burden following OrfV therapy revealed an optimal re-administration time to sustain antitumor immunity, further extending survival. The data presented highlight the importance of investigating immune response kinetics following immunotherapy and demonstrate that detailed kinetic profiling of immune responses can reveal novel insights into mechanisms of action that can guide the development of more effective therapies.

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.

Prognostic value of epigenetic markers for canine mast cell cancer.

Canine Mast cell tumors (MCTs) constitute approximately 21% of all canine skin tumors. Despite the use of comprehensive grading systems, biological aggressiveness is sometimes difficult to predict, therefore there is a need for better prognostic markers. Progression in various cancers involves DNA hypermethylation, hypomethylation and epigenetic enzyme dysregulation. Therefore, global levels of 5-methylcytosine, 5-hydroxymethylcytosine and associated enzymes DNMT1, and IDH1 expression may predict MCT aggressiveness. A tissue microarray (TMA) with cores from 244 different tumor samples from 189 dogs was immunolabelled and used to quantify the global DNA methylation and hydroxymethylation levels as well as the levels of the enzymes involved in DNA methylation and their relationship with canine MCT outcome. From the immunolabelled TMA, H-scores were generated using QuPath (v0.1.2) and analyzed with associated patient data. High 5MC and DNMT1, and low IDH1 levels were associated with poorer outcome when looking at all canine MCT cases. High 5MC levels showed significance for shorter disease-free interval (DFI) in subcutaneous cases and high 5MC levels showed poorer DFI and overall survival (OS) in cases with Kiupel's grading system high grade. Cases with grade II in Patnaik's grading system showed better DFI with low levels of DNMT1 and better OS with low levels of 5MC and 5HMC. High levels of DNMT1 staining were also associated with shorter DFI for dermal MCTs. For cases that received adjuvant therapy in addition to surgery, all parameters except IDH1 were significantly associated with OS. Therefore, there is potential for DNA methylation status and levels of enzymes associated with DNA methylation pathways to better predict outcome in canine MCT, and to possibly influence treatment decisions.

Neutrophils in Dendritic Cell-Based Cancer Vaccination: The Potential Roles of Neutrophil Extracellular Trap Formation.

Neutrophils have conflicting roles in the context of cancers, where they have been associated with contributing to both anti-tumor and pro-tumor responses. Their functional heterogenicity is plastic and can be manipulated by environmental stimuli, which has fueled an area of research investigating therapeutic strategies targeting neutrophils. Dendritic cell (DC)-based cancer vaccination is an immunotherapy that has exhibited clinical promise but has shown limited clinical efficacy. Enhancing our understanding of the communications occurring during DC cancer vaccination can uncover opportunities for enhancing the DC vaccine platform. There have been observed communications between neutrophils and DCs during natural immune responses. However, their crosstalk has been poorly studied in the context of DC vaccination. Here, we review the dual functionality of neutrophils in the context of cancers, describe the crosstalk between neutrophils and DCs during immune responses, and discuss their implications in DC cancer vaccination. This discussion will focus on how neutrophil extracellular traps can influence immune responses in the tumor microenvironment and what roles they may play in promoting or hindering DC vaccine-induced anti-tumor efficacy.

Multiplex flow cytometry-based assay for quantifying tumor- and virus-associated antibodies induced by immunotherapies.

Novel immunotherapies continue to be developed and tested for application against a plethora of diseases. The clinical translation of immunotherapies requires an understanding of their mechanisms. The contributions of antibodies in driving long-term responses following immunotherapies continue to be revealed given their diverse effector functions. Developing an in-depth understanding of the role of antibodies in treatment efficacy is required to optimize immunotherapies and improve the chance of successfully translating them into the clinic. However, analyses of antibody responses can be challenging in the context of antigen-agnostic immunotherapies, particularly in the context of cancers that lack pre-defined target antigens. As such, robust methods are needed to evaluate the capacity of a given immunotherapy to induce beneficial antibody responses, and to identify any therapy-limiting antibodies. We previously developed a comprehensive method for detecting antibody responses induced by antigen-agnostic immunotherapies for application in pre-clinical models of vaccinology and cancer therapy. Here, we extend this method to a high-throughput, flow cytometry-based assay able to identify and quantify isotype-specific virus- and tumor-associated antibody responses induced by immunotherapies using small sample volumes with rapid speed and high sensitivity. This method provides a valuable and flexible protocol for investigating antibody responses induced by immunotherapies, which researchers can use to expand their analyses and optimize their own treatment regimens.

Feline Oncogenomics: What Do We Know about the Genetics of Cancer in Domestic Cats?

Cancer is a significant cause of morbidity and mortality in domestic cats. In humans, an understanding of the oncogenome of different cancer types has proven critical and is deeply interwoven into all aspects of patient care, including diagnostics, prognostics and treatments through the application of targeted therapies. Investigations into understanding the genetics of feline cancers started with cytogenetics and was then expanded to studies at a gene-specific level, looking for mutations and expression level changes of genes that are commonly mutated in human cancers. Methylation studies have also been performed and together with a recently generated high-quality reference genome for cats, next-generation sequencing studies are starting to deliver results. This review summarises what is currently known of the genetics of both common and rare cancer types in cats, including lymphomas, mammary tumours, squamous cell carcinomas, soft tissue tumours, mast cell tumours, haemangiosarcomas, pulmonary carcinomas, pancreatic carcinomas and osteosarcomas. Shining a spotlight on our current understanding of the feline oncogenome will hopefully serve as a springboard for more much-needed research into the genetics of cancer in domestic cats.

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.

Proteomic Assessment of Extracellular Vesicles from Canine Tissue Explants as a Pipeline to Identify Molecular Targets in Osteosarcoma: PSMD14/Rpn11 as a Proof of Principle.

Osteosarcoma (OS) is a highly malignant bone tumour that has seen little improvement in treatment modalities in the past 30 years. Understanding what molecules contribute to OS biology could aid in the discovery of novel therapies. Extracellular vesicles (EVs) serve as a mode of cell-to-cell communication and have the potential to uncover novel protein signatures. In our research, we developed a novel pipeline to isolate, characterize, and profile EVs from normal bone and osteosarcoma tissue explants from canine OS patients. Proteomic analysis of vesicle preparations revealed a protein signature related to protein metabolism. One molecule of interest, PSMD14/Rpn11, was explored further given its prognostic potential in human and canine OS, and its targetability with the drug capzimin. In vitro experiments demonstrated that capzimin induces apoptosis and reduces clonogenic survival, proliferation, and migration in two metastatic canine OS cell lines. Capzimin also reduces the viability of metastatic human OS cells cultured under 3D conditions that mimic the growth of OS cells at secondary sites. This unique pipeline can improve our understanding of OS biology and identify new prognostic markers and molecular targets for both canine and human OS patients.

RNA-Seq analysis of gene expression in 25 cases of canine lymphoma undergoing CHOP chemotherapy.

OBJECTIVES: Canine lymphoma, the most common hematological cancer in dogs, shares many molecular and clinical characteristics with human Non-Hodgkin lymphoma (NHL). The standard treatment for canine lymphoma is "CHOP" multiagent chemotherapy protocol consisting of Cyclophosphamide, Doxorubicin (Hydroxydaunorubicin), Vincristine (Oncovin™), and Prednisone. Approximately 70-85% of patients treated with CHOP achieve clinical remission. However, duration of remission varies and the majority of dogs eventually relapse. To identify possible biomarkers for patients failing to achieve remission, we performed RNA-Seq analysis on 25 cases of canine lymphoma obtained prior the start of their CHOP therapy regime and assessed gene expression associated with patient progression free survival (PFS). DATA DESCRIPTION: The data consists of (1) raw RNA-Seq reads in 75 bp fastq format from fine needle aspirate samples of enlarged lymph nodes from canine patients with naturally occurring lymphoma; (2) Fragments Per Kilobase Million (FPKM) values for each sample; (3) raw transcript counts for each sample; (4) anonymized patient details including PFS; (5) heat map of gene expression and (6) Cox proportional hazard analysis showing significantly expressed genes. These data may be useful for comparative analysis of gene expression in human NHL and analysis of gene expression associated with disease outcome in canine lymphoma.

Histological and histochemical characteristics of lacrimal glands in beluga whales (Delphinapterus leucas).

The objective of this study was to describe the histological and histochemical characteristics of the lacrimal glands of beluga whales. The study was carried out on the formalin-fixed ocular globes from 96 carcasses of beluga whales found stranded in the St. Lawrence estuary in Quebec, Canada. Hematoxylin and eosin (H&E) stained slides from the eyes of each whale were examined for lacrimal glands. Histological description was done with H&E and Masson Trichrome (MT) stains. Period Acid-Schiff (PAS), Alcian blue (AB) pH 1.0 and 2.5, and High Iron Diamine (HID) stains were used for histochemical characterization of glycoproteins. Thirteen ocular samples from animals ranging from neonate to 48 y included sections of lacrimal glands. The H&E stain revealed a tubuloalveolar gland architecture, separated into lobules by dense connective tissue. Each lobule contained a mixture of acini and tubules with ductules. Small and large acini were composed of low and tall columnar cells, respectively. Acinar cells contained basophilic cytoplasmic granules. The ductules were lined with a bi-layered cuboidal-to-squamous epithelium. The MT stain highlighted the connective tissue separating ductules and acini. Large acini were positive for PAS and some small acini had patchy uptake. Positive staining for AB pH 1.0 and 2.5 was mainly seen in tall columnar cells as compared to small acini that had faint to no stain uptake. High Iron Diamine stain revealed 90% staining of all acinar cells, with 10% exhibiting a mixed blue-black tinge. It was concluded that the lacrimal glands of beluga whales have similar histological and histochemical findings to those of artiodactyla and carnivora orders.

L'objectif de cette étude était de décrire les caractéristiques histologiques et histochimiques des glandes lacrymales des bélugas. L'étude a été réalisée sur les globes oculaires fixés au formol de 96 carcasses de bélugas trouvées échouées dans l'estuaire du Saint-Laurent au Québec, Canada. Des lames colorées à l'hématoxyline et à l'éosine (H&E) des yeux de chaque baleine ont été examinées pour la présence de glandes lacrymales. La description histologique a été réalisée avec des colorations H&E et trichrome de Masson (MT). Les colorations Periodic acid-Schiff (PAS), au bleu Alcian (AB) pH 1,0 et 2,5, et diamine à haute teneur en fer (HID) ont été utilisées pour la caractérisation histochimique des glycoprotéines. Treize échantillons oculaires provenant d'animaux allant du nouveau-né à 48 ans comprenaient des sections de glandes lacrymales. La coloration H&E a révélé une architecture de glande tubulo-alvéolaire, séparée en lobules par un tissu conjonctif dense. Chaque lobule contenait un mélange d'acini et de tubules avec des ductules. Les petits et les grands acini étaient respectivement composés de cellules cylindriques basses et hautes. Les cellules acinaires contenaient des granules cytoplasmiques basophiles. Les canaux étaient tapissés d'un épithélium cuboïde à squameux bicouche. La coloration MT a mis en évidence le tissu conjonctif séparant les canaux et les acini. Les grands acini étaient positifs pour le PAS et certains petits acini avaient une absorption inégale. Une coloration positive pour AB pH 1,0 et 2,5 a été principalement observée dans les cellules cylindriques hautes par rapport aux petits acini qui avaient une absorption de coloration faible ou nulle. La coloration HDI a révélé une coloration de 90 % de toutes les cellules acinaires, 10 % présentant une teinte mixte bleu-noir. Il a été conclu que les glandes lacrymales des bélugas présentent des résultats histologiques et histochimiques similaires à ceux des ordres des artiodactyles et des carnivores.(Traduit par Docteur Serge Messier).

Beclin-1 is a novel predictive biomarker for canine cutaneous and subcutaneous mast cell tumors.

Mast cell tumors (MCTs) are the most common skin tumor of the dog, and accurately predicting their clinical behavior is critical in directing patient therapy, as they range from benign lesions to a fatal systemic disease. Grading is useful for prognosis, but it cannot predict the behavior of all MCTs. We hypothesized that biomarker immunolabeling in tumor tissues would correlate with patient morbidity and mortality. A clinically annotated tissue microarray (TMA) of primary, recurrent, and metastatic (to lymph node) canine dermal and subcutaneous MCTs was created. Some dogs whose MCTs were included in the TMA did not receive adjunctive treatment after surgical excision of the MCT, whereas others were treated with one or a combination of chemotherapy, radiation, or oral toceranib. Immunohistochemistry for beclin-1, an autophagy protein, was performed followed by digital image analysis. Beclin-1 immunolabeling was higher in recurrent tumors (mean H-score 110.8) than primary MCTs (mean H-score 73.5), and highest in lymph node metastases (mean H-score 138.5) with a significant difference in means (P < .001). While beclin-1 level was not prognostic, it was strongly predictive for survival after adjunctive treatment; dogs with high beclin-1-expressing tumors showed poorer survival compared to those with low beclin-1-expressing tumors (HR = 5.7, P = .02), especially in Kiupel high-grade tumors (HR = 16.3, P = .01). Beclin-1 immunolabeling was the only significant predictive factor by multivariable analysis (P = .04). These findings may improve our ability to predict the response to adjunctive therapy. Importantly, these data suggest that autophagy inhibitors may be useful in improving response to treatment for dogs with high-grade MCTs.

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.

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.

Dendritic Cell-Based Vaccines Recruit Neutrophils to the Local Draining Lymph Nodes to Prime Natural Killer Cell Responses.

Dendritic cell (DC)-based cancer vaccines are a form of immunotherapy that activates the innate and adaptive immune systems to combat cancers. Neutrophils contribute to cancer biology and have the potential to be exploited by immunotherapeutic platforms to enhance anti-tumor immune responses. We previously showed that DC vaccines elicit the expansion of mouse interferon (IFN)γ-producing mature natural killer (NK) cells to elevate anti-tumor responses. Here, we demonstrate the rapid recruitment of neutrophils to the draining lymph nodes of DC-vaccinated mice. This was accompanied by an increase in the total number of NK cells producing IFNγ and expressing CD107a, a marker of degranulation that demonstrates NK cell functional activity. Furthermore, the depletion of neutrophils in DC-immunized mice resulted in decreased numbers of NK cells in draining lymph nodes compared to the controls. Interestingly, the increased number of IFNγ- and CD107a-expressing NK cells in DC-immunized mice was not detected in mice depleted of neutrophils. Further investigations showed that DC vaccines induced IFNγ- and TNFα-producing CD8+ T cells that also expressed CD107a, but depletion of neutrophils did not have any impact on the CD8+ T cell population. Our findings suggest that neutrophil-mediated anti-tumor immunity induced by a DC vaccine platform could be targeted to provide innovative strategies to enhance its clinical efficacy.

Unilateral primary carcinoma of the kidney with central nervous system invasion and vertebral lysis in a cat.

Case summary: A young adult female spayed domestic shorthair cat presented for acute hindlimb weakness and anorexia with a 1-month history of lethargy, hyporexia and weight loss. A mass was palpable in the caudolateral abdomen and the left hindlimb was diffusely edematous. Abdominal ultrasound showed hydronephrosis of the left kidney with suspected hydroureter and heterogeneous tissue in the dorsal abdomen. CT evaluation confirmed a mass extending from the left kidney through the lumbar musculature with hydronephrosis, aortic attenuation, caudal vena caval thrombosis and lysis of vertebrae 4 and 5. Fine-needle aspiration of the mass suggested squamous cell carcinoma. Owing to clinical deterioration, euthanasia was elected. At necropsy, the left kidney was firmly adhered to the lumbar region with tissue that obliterated the musculature and surrounded the aorta and vena cava. There was hydronephrosis of the left kidney. Histopathologic evaluation of the mass revealed islands of neoplastic epithelial cells separated by fibrous connective tissue and areas of gradual keratinization with rare squamous metaplasia. The histologic diagnosis was invasive carcinoma with desmoplasia and vascular invasion. Relevance and novel information: Primary carcinomas of the kidney in cats are rare and this report documents a progression of disease not previously reported in cats. This is the second reported case of a primary carcinoma of renal origin with features of squamous cell carcinoma in a cat, and the first with lumbar and vascular invasion. This is also the first use of kidney injury molecule-1 to help investigate tumor differentiation in cats.

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.

Recent Advances in the Discovery of Biomarkers for Canine Osteosarcoma.

Canine osteosarcoma (OSA) is an aggressive malignancy that frequently metastasizes to the lung and bone. Not only has there been essentially no improvement in therapeutic outcome over the past 3 decades, but there is also a lack of reliable biomarkers in clinical practice. This makes it difficult to discriminate which patients will most benefit from the standard treatment of amputation and adjuvant chemotherapy. The development of reliable diagnostic biomarkers could aid in the clinical diagnosis of primary OSA and metastasis; while prognostic, and predictive biomarkers could allow clinicians to stratify patients to predict response to treatment and outcome. This review summarizes biomarkers that have been explored in canine OSA to date. The focus is on molecular biomarkers identified in tumor samples as well as emerging biomarkers that have been identified in blood-based (liquid) biopsies, including circulating tumor cells, microRNAs, and extracellular vesicles. Lastly, we propose future directions in biomarker research to ensure they can be incorporated into a clinical setting.