High-Grade, Stage 2 Mast Cell Tumors: Outcome in Dogs With Local and Systemic Therapy.

Canine mast cell tumors (MCTs) have highly variable clinical behavior, and predicting outcomes in individual dogs remains challenging. Many studies combine dogs with varying tumor grades, clinical stage, or treatments, confounding those results. The purpose of this retrospective study was to determine outcome and prognostic factors in a specific subset of dogs with high-grade, stage 2, cutaneous MCTs treated with adequate local control via surgery with or without radiation therapy and adjuvant cytotoxic chemotherapy. Seventeen dogs met the inclusion criteria, and the median survival time was 259 days. Development of local recurrence, tumor location, and presence of ulceration were all associated with shorter survival times. Tumor size, mitotic count, chemotherapy protocol, lymph node classification, and radiation therapy were not significantly associated with outcome. In this study, a specific population of dogs characterized by high-grade MCTs with local lymph node metastasis who received aggressive local and systemic therapy had a median survival of about 8.5 mo. Dogs with ulcerated tumors, recurrent tumors, or tumors located on the head had a worse outcome despite aggressive therapy. These results may serve as a basis of comparison for future research exploring alternative treatment combinations in this specific population of dogs.

MRI characteristics of canine prostatic neoplasia.

Magnetic resonance imaging (MRI) has been used to evaluate dogs with suspected prostatic neoplasia, however, published studies describing MRI characteristics of canine prostatic neoplasia are currently lacking. The aims of the current retrospective case series study were to describe MRI findings of the pelvic region in dogs with a histopathologic or cytologic diagnosis of prostatic neoplasia. Retrospective analysis of these images was then performed by a board-certified veterinary radiologist for shared imaging characteristics. The most consistent characteristics were heterogeneous hyperintensity of the tumor on T2-weighted images (10/10) and short tau inversion recovery images (10/10), prostatic capsular margin distortion by the tumor (10/10), cavitations (10/10), complete effacement of the prostatic architecture (9/10), neurovascular bundle (NVB) compression or invasion (9/10), heterogeneous isointensity of the tumor on T1-weighted images (9/10), and strong contrast enhancement of the tumor (8/10). Additional features included an overlying pattern of distorted radiating striations (7/10), regional lymphadenomegaly (5/10), mineralization within the mass (5/10), urinary bladder trigone involvement (6/10), and post-prostatic urethral involvement (7/10). These findings supported the use of MRI as an adjunct imaging modality for diagnosis and therapeutic planning of prostatic neoplasia and including prostatic neoplasia as a likely differential diagnosis for dogs with these MRI characteristics.

Whole genome variant association across 100 dogs identifies a frame shift mutation in DISHEVELLED 2 which contributes to Robinow-like syndrome in Bulldogs and related screw tail dog breeds.

Domestic dog breeds exhibit remarkable morphological variations that result from centuries of artificial selection and breeding. Identifying the genetic changes that contribute to these variations could provide critical insights into the molecular basis of tissue and organismal morphogenesis. Bulldogs, French Bulldogs and Boston Terriers share many morphological and disease-predisposition traits, including brachycephalic skull morphology, widely set eyes and short stature. Unlike other brachycephalic dogs, these breeds also exhibit vertebral malformations that result in a truncated, kinked tail (screw tail). Whole genome sequencing of 100 dogs from 21 breeds identified 12.4 million bi-allelic variants that met inclusion criteria. Whole Genome Association of these variants with the breed defining phenotype of screw tail was performed using 10 cases and 84 controls and identified a frameshift mutation in the WNT pathway gene DISHEVELLED 2 (DVL2) (Chr5: 32195043_32195044del, p = 4.37 X 10-37) as the most strongly associated variant in the canine genome. This DVL2 variant was fixed in Bulldogs and French Bulldogs and had a high allele frequency (0.94) in Boston Terriers. The DVL2 variant segregated with thoracic and caudal vertebral column malformations in a recessive manner with incomplete and variable penetrance for thoracic vertebral malformations between different breeds. Importantly, analogous frameshift mutations in the human DVL1 and DVL3 genes cause Robinow syndrome, a congenital disorder characterized by similar craniofacial, limb and vertebral malformations. Analysis of the canine DVL2 variant protein showed that its ability to undergo WNT-induced phosphorylation is reduced, suggesting that altered WNT signaling may contribute to the Robinow-like syndrome in the screwtail breeds.

Retrospective analysis of doxorubicin and prednisone as first-line therapy for canine B-cell lymphoma.

BACKGROUND: A doxorubicin (DOX)-based chemotherapy protocol, CHOP, is the most effective treatment for canine high-grade B-cell lymphoma; however, the cost and time requirements associated with this protocol are not feasible for many pet owners. An alternative treatment option is the use of DOX, the most effective drug, in combination with prednisone. Prior studies with single-agent DOX included dogs with T-cell lymphoma, a known negative prognostic factor, which may have resulted in shorter reported survival times than if dogs with B-cell lymphoma were analyzed separately. The purpose of this study was to evaluate the outcome of dogs with high-grade B-cell lymphoma when treated with DOX and prednisone with or without L-asparaginase (L-ASP). Identification of prognostic factors was of secondary interest. RESULTS: Thirty-three dogs were included in the study; 31 dogs were evaluable for response with an overall response rate of 84%. The median progression free survival (PFS) and overall survival (OS) were 147 days and 182 days, respectively. The one-year survival fraction was 23%. No variable other than protocol completion was found to be significant for either PFS or OS including historical prognostic factors such as substage, thrombocytopenia, and body weight. CONCLUSIONS: Dogs with high-grade B-cell lymphoma treated with DOX and prednisone with or without L-ASP have similar response rates, PFS, and OS to prior studies that did not differentiate between lymphoma immunophenotype. This protocol is not a replacement for CHOP; however, it is an alternative if time and cost are factors, while providing therapeutic benefit greater than prednisone alone.

Management of an invasive and metastatic Sertoli cell tumor with associated myelotoxicosis in a dog.

We describe the surgical and post-operative management of a large, invasive, and metastatic functional Sertoli cell tumor in a 9-year-old cryptorchid male Labrador retriever dog. Despite residual disease after surgery, bone marrow recovery occurred without administration of bone marrow stimulants and serum estradiol accurately predicted tumor recurrence.

Gestion d'une tumeur à cellules de Sertoli invasive et métastatique avec une myélotoxicose secondaire chez un chien. Nous décrivons la gestion chirurgicale et postopératoire d'une tumeur à cellules de Sertoli fonctionnelles, de grande taille, invasive et métastatique chez un chien Labrador retriever cryptorchide âgé de 9 ans. Malgré une maladie résiduelle après la chirurgie, le rétablissement de la moelle osseuse s'est produit sans l'administration de stimulants de la moelle osseuse et l'œstradiol sérique a fidèlement prédit la récurrence de la tumeur.(Traduit par Isabelle Vallières).

Epigenetic regulation of human hedgehog interacting protein in glioma cell lines and primary tumor samples.

Glioma constitutes one of the most common groups of brain tumors, and its prognosis is influenced by different genetic and epigenetic modulations. In this study, we demonstrated low or no expression of hedgehog interacting protein (HHIP) in most of the cell lines and primary glioma tumor samples. We further proceeded to promoter methylation study of this gene in the same cell lines and primary tumor samples and found 87 % (7/8) HHIP methylation in glioblastoma cell lines and 75 % (33/44) in primary tumor samples. These methylation pattern correlates with low or unexpressed HHIP in both cell lines and primary tumor samples. Our results suggest the possibility of epigenetic regulation of this gene in glioma, similarly to medulloblastoma, gastric, hepatic, and pancreatic cancers. Also, HHIP might be a diagnostic or prognostic marker in glioma and help to the detection of these tumors in early stages of disease.

Unilateral intraocular mastocytosis and anterior uveitis in a dog with subcutaneous mast cell tumors.

A 9-year-old male castrated Scottish terrier was referred to the Radiation Oncology Service at the William R. Pritchard Veterinary Medical Teaching Hospital for palliative radiation therapy of an incompletely excised, recurrent subcutaneous mast cell tumor (MCT) located over the right scapula, and surgical removal of a perianal MCT. Three weeks after initial presentation and prior to the fifth radiation treatment, the patient was presented with cloudiness of the left eye of 3-7 days duration. Ophthalmic consultation revealed 3+ aqueous flare with a dependent, swirling component filling approximately one-third of the anterior chamber. Aqueocentesis was performed under general anesthesia. Cytology revealed mast cells with highly atypical morphology and considered most consistent with neoplasia. The patient died 7 months after pathologic diagnosis of MCT on the right shoulder and 2 months after the cytologic diagnosis of malignant mast cells in the left anterior chamber. To the authors' knowledge, this is the first report of intraocular involvement in a mammal with MCTs, described here as intraocular mastocytosis.

Improved characterization and translation of NK cells for canine immunotherapy.

The field of cancer immunology has seen a meteoric rise in interest and application due to the discovery of immunotherapies that target immune cells, often leading to dramatic anti-tumor effects. However, successful cellular immunotherapy for solid tumors remains a challenge, and the application of immunotherapy to dogs with naturally occurring cancers has emerged as a high yield large animal model to bridge the bench-to-bedside challenges of immunotherapies, including those based on natural killer (NK) cells. Here, we review recent developments in the characterization and understanding of canine NK cells, a critical springboard for future translational NK immunotherapy research. The characterization of canine NK cells is exceptionally pertinent given the ongoing challenges in defining them and contextualizing their similarities and differences compared to human and murine NK cells compounded by the limited availability of validated canine specific reagents. Additionally, we summarize the current landscape of the clinical and translational literature employing strategies to capitalize on endogenous and exogenous NK cell immunotherapy in canine cancer patients. The insights regarding efficacy and immune correlates from these trials provide a solid foundation to design and test novel combinational therapies to enhance NK cell activity with the added benefit of motivating comparative work to translate these findings to human cancers with extensive similarities to their canine counterparts. The compilation of knowledge from basic canine NK phenotype and function to applications in first-in-dog clinical trials will support the canine cancer model and enhance translational work to improve cancer outcomes for both dogs and humans.

A variant in the 5'UTR of ERBB4 is associated with lifespan in Golden Retrievers.

Genome-wide association studies (GWAS) in long-lived human populations have led to identification of variants associated with Alzheimer's disease and cardiovascular disease, the latter being the most common cause of mortality in people worldwide. In contrast, naturally occurring cancer represents the leading cause of death in pet dogs, and specific breeds like the Golden Retriever (GR) carry up to a 65% cancer-related death rate. We hypothesized that GWAS of long-lived GRs might lead to the identification of genetic variants capable of modifying longevity within this cancer-predisposed breed. A GWAS was performed comparing GR dogs ≥ 14 years to dogs dying prior to age 12 which revealed a significant association to ERBB4, the only member of the epidermal growth factor receptor family capable of serving as both a tumor suppressor gene and an oncogene. No coding variants were identified, however, distinct haplotypes in the 5'UTR were associated with reduced lifespan in two separate populations of GR dogs. When all GR dogs were analyzed together (n = 304), the presence of haplotype 3 was associated with shorter survival (11.8 years vs. 12.8 years, p = 0.024). GRs homozygous for haplotype 3 had the shortest survival, and GRs homozygous for haplotype 1 had the longest survival (11.6 years vs. 13.5 years, p = 0.0008). Sub-analyses revealed that the difference in lifespan for GRs carrying at least 1 copy of haplotype 3 was specific to female dogs (p = 0.009), whereas survival remained significantly different in both male and female GRs homozygous for haplotype 1 or haplotype 3 (p = 0.026 and p = 0.009, respectively). Taken together, these findings implicate a potential role for ERBB4 in GR longevity and provide evidence that within-breed canine lifespan studies could serve as a mechanism to identify favorable or disease-modifying variants important to the axis of aging and cancer.

Preclinical evaluation and first-in-dog clinical trials of PBMC-expanded natural killer cells for adoptive immunotherapy in dogs with cancer.

BACKGROUND: Natural killer (NK) cells are cytotoxic cells capable of recognizing heterogeneous cancer targets without prior sensitization, making them promising prospects for use in cellular immunotherapy. Companion dogs develop spontaneous cancers in the context of an intact immune system, representing a valid cancer immunotherapy model. Previously, CD5 depletion of peripheral blood mononuclear cells (PBMCs) was used in dogs to isolate a CD5dim-expressing NK subset prior to co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. This study aimed to assess NK activation, expansion, and preliminary clinical activity in first-in-dog clinical trials using a novel system with unmanipulated PBMCs to generate our NK cell product. METHODS: Starting populations of CD5-depleted cells and PBMCs from healthy beagle donors were co-cultured for 14 days, phenotype, cytotoxicity, and cytokine secretion were measured, and samples were sequenced using the 3'-Tag-RNA-Seq protocol. Co-cultured human PBMCs and NK-isolated cells were also sequenced for comparative analysis. In addition, two first-in-dog clinical trials were performed in dogs with melanoma and osteosarcoma using autologous and allogeneic NK cells, respectively, to establish safety and proof-of-concept of this manufacturing approach. RESULTS: Calculated cell counts, viability, killing, and cytokine secretion were equivalent or higher in expanded NK cells from canine PBMCs versus CD5-depleted cells, and immune phenotyping confirmed a CD3-NKp46+ product from PBMC-expanded cells at day 14. Transcriptomic analysis of expanded cell populations confirmed upregulation of NK activation genes and related pathways, and human NK cells using well-characterized NK markers closely mirrored canine gene expression patterns. Autologous and allogeneic PBMC-derived NK cells were successfully expanded for use in first-in-dog clinical trials, resulting in no serious adverse events and preliminary efficacy data. RNA sequencing of PBMCs from dogs receiving allogeneic NK transfer showed patient-unique gene signatures with NK gene expression trends in response to treatment. CONCLUSIONS: Overall, the use of unmanipulated PBMCs appears safe and potentially effective for canine NK immunotherapy with equivalent to superior results to CD5 depletion in NK expansion, activation, and cytotoxicity. Our preclinical and clinical data support further evaluation of this technique as a novel platform for optimizing NK immunotherapy in dogs.

Treatment of retroperitoneal sarcoma results in improved outcomes.

OBJECTIVE: To report the clinical characteristics, treatments, and outcomes in a cohort of dogs with histologically confirmed retroperitoneal sarcoma (RPS) and to identify potential variables of prognostic significance. ANIMALS: 46 client-owned dogs from 10 clinics with histopathologic diagnosis of a sarcoma originating from the retroperitoneal space. METHODS: Medical records were retrospectively reviewed to obtain information regarding clinical characteristics, treatments, and outcomes. Recorded variables were analyzed to report descriptive data for all cases and overall survival time. Multivariate analysis was utilized to evaluate prognostic factors for overall survival. RESULTS: Hemangiosarcoma was the most common histologic subtype diagnosed (76.1%). Cytoreductive and curative intent surgical excision of the RPS was attempted in 12 and 22 dogs, respectively; 12 dogs underwent no surgery or had an exploratory laparotomy with incisional biopsy only. Nineteen dogs received adjuvant chemotherapy, either injectable or metronomic, and 1 dog received adjuvant radiation therapy. Fourteen of the 34 (41.2%) surgically treated dogs developed evidence of local recurrence, but there was no difference in local recurrence when comparing dogs categorized as curative intent versus cytoreductive surgery. The median overall survival time was 238 days. On multivariable analysis, treatment approach was associated with survival with surgical excision (vs palliative treatment) and adjuvant chemotherapy following surgery being protective against death. A diagnosis of hemangiosarcoma was associated with a greater hazard of death. CLINICAL RELEVANCE: This study demonstrates a substantially greater survival time than previously published and suggests a survival benefit from surgical excision and adjuvant chemotherapy.

Efficacy and toxicity of carboplatin in the treatment of macroscopic mesenchymal neoplasia in dogs.

Palliative chemotherapy options for dogs with macroscopic non-osseous mesenchymal tumours are limited. The purpose of this study was to assess the response rate of these tumours to carboplatin chemotherapy. Medical records of 28 dogs treated with carboplatin for macroscopic mesenchymal neoplasia between 1990 and 2022 were retrospectively reviewed. Sixteen dogs with soft tissue sarcoma and 12 dogs with haemangiosarcoma were included. Responses observed included one complete response and three partial responses, for an overall response rate of 14.2% (4/28) and median time to progression of 42 days (range 21-259 days). Responses were only seen in patients with haemangiosarcoma, for a response rate of 33.3% (4/12) and median time to progression for responders of 103 days (range 39-252 days). Median time to progression for dogs with metastatic disease was similar to those with only local disease (distant median: 44 days; local median: 23 days, p = 0.56). Dogs with chemotherapy-naïve disease were compared to dogs having received previous chemotherapy treatment and had a median time to progression of 75 days and 40.5 days respectively (p = 0.13). Twenty-two dogs experienced 48 adverse events, with most being grade 1 or 2 (79%). Carboplatin was well tolerated, with variable macroscopic anti-tumour activity and short response duration. Carboplatin may be an acceptable rescue option for dogs with macroscopic haemangiosarcoma, especially those patients that cannot receive doxorubicin.

Missing a "Missing Self" Mechanism: Modeling and Detection of Ly49 Expression in Canine NK Cells.

NK cells are a key focus in immuno-oncology, based on their ability to eliminate malignant cells without prior sensitization. Dogs are valuable models for translational immunotherapy studies, especially for NK cells, where critical species differences exist between mice and humans. Given that the mechanism for recognition of "self" by canine NK cells is currently unknown, we sought to evaluate expression of Ly49 in canine NK cells using in silico and high-throughput techniques. We interrogated the identified polymorphism/mutation in canine Ly49 and assessed the potential impact on structure using computational modeling of three-dimensional protein structure and protein-protein docking of canine Ly49 with MHC class I (MHC-I). Bulk and single-cell RNA-sequencing analysis was performed to detect gene expression of Ly49/KLRA1 in resting and activated NK cells. Tertiary protein structure demonstrated significant structural similarity to the known murine system. Molecular docking of canine Ly49 with MHC-I was favorable, converging at a single low-energy conformation. RNA sequencing revealed expression of Ly49/KLRA1 in both resting and activated NK cells and demonstrated almost exclusive expression of the gene in the NK cluster at the single-cell level. Despite prior reports of a mutated, nonfunctional canine Ly49, our data support that the protein product is predicted to bind to MHC-I in a comparable conformation to the murine system and is expressed in canine NK cells with upregulation following activation. Taken together, these data suggest that Ly49 is capable of recognizing MHC-I and therefore regulating NK cell function in dogs.

G2/M arrest and mitotic slippage induced by fenbendazole in canine melanoma cells.

BACKGROUND: The use of fenbendazole (FBZ) in terminal cancer patients has recently increased, as anthelminthic drugs, such as FBZ and benzimidazole, exhibit anti-tubulin effects in tumour cells. OBJECTIVES: The present study evaluated the in vitro anti-cancer effects of FBZ in five canine melanoma cell lines originating from the oral cavity (UCDK9M3, UCDK9M4, UCDK9M5, KMeC and LMeC). METHODS: Five canine melanoma cell lines were treated with FBZ and analysed with cell viability assay, cell cycle analysis, western blot assay and immunofluorescence staining to identify apoptotic effect, cell cycle arrest, microtubule disruption and mitotic slippage. RESULTS: Cell viability was reduced in all melanoma cell lines in a dose-dependent manner after FBZ treatment. Through cell cycle analysis, G2/M arrest and mitotic slippage were identified, which showed a time-dependent change. All treatment concentrations induced increased cleaved PARP signals in western blot analysis compared to the control groups. Immunofluorescence of cells treated for 24 h revealed defects in microtubule structure, multinucleation or macronucleation. With the exception of UCDK9M3, the melanoma cells showed mitotic slippage and post-slippage death, indicative of mitotic catastrophe. CONCLUSIONS: These results indicate that FBZ exhibits anti-cancer effects in vitro against canine melanoma cells; however, further in vivo studies regarding the clinical applications of FBZ are required.

Expression and therapeutic targeting of BMI1 in canine gliomas.

The BMI1 proto-oncogene, polycomb ring finger protein (BMI1) is a key component of the epigenetic polycomb repressor complex 1, and has been associated with aggressive behaviour and chemotherapeutic resistance in various malignances including human gliomas. Similar to humans, spontaneous canine gliomas carry a poor prognosis with limited therapeutic options. BMI1 expression and the effects of BMI1 inhibition have not been evaluated in canine gliomas. Here, we demonstrate that BMI1 is highly expressed in canine gliomas. Although increased BMI1 protein expression correlated with higher glioma grade in western blot assays, this correlation was not observed in a larger sample set using immunohistochemical analysis. The BMI1 inhibitor, PTC-209, suppressed BMI1 expression in established canine glioma cell lines and resulted in antiproliferative activity when used alone and in combination with chemotherapeutic agents. PTC-209 targeting of BMI1 activated the retinoblastoma (RB) pathway through downregulation of total and phosphorylated RB, independent of INK4A/ARF signalling, likely through BMI1-inhibition mediated upregulation of p21. These data support the rationale for targeting of BMI1 signalling and the use of canine glioma as a translational therapeutic model for human disease.

Expression of the hedgehog signalling pathway and the effect of inhibition at the level of smoothened in canine osteosarcoma cell lines.

Osteosarcoma (OSA) is the most common malignant bone cancer in dogs. Canine and human OSA share several features, including tumour environments, response to traditional treatment, and several molecular pathways. Hedgehog (Hh) signalling is known to contribute to tumorigenesis and progression of various cancers, including human OSA. This study aimed to identify the role of the Hh signalling pathway in canine OSA cell lines, including Abrams, D17, and Moresco, focusing on the signal transducer Smoothened (SMO). mRNA and protein levels of Hh pathway components, including SHH, IHH, SMO, and PTCH1, were aberrant in all examined OSA cell lines compared with canine osteoblast cells. The SMO inhibitor cyclopamine significantly decreased cell viability and colony-forming ability in the canine OSA cell lines in a dose-dependent manner. Moresco cells, which expressed the highest level of SMO protein, were the most sensitive to the anticancer effect of cyclopamine among the three canine OSA cell lines tested. Hh downstream target gene and protein expression in canine OSA cell lines were downregulated after cyclopamine treatment. In addition, cyclopamine significantly increased apoptotic cell death in Abrams and Moresco cells. The findings that Hh/SMO is activated in canine OSA cell lines and cyclopamine suppresses OSA cell survival via inhibition of SMO suggest that the Hh/SMO signalling pathway might be a novel therapeutic target for canine OSA.

Retrospective immunohistochemical investigation of suspected non-visceral leiomyosarcoma in dogs.

Visceral leiomyosarcoma is well described in dogs, but information about non-visceral locations and prevalence is lacking. The diagnosis of leiomyosarcoma is challenging without a gold standard, and often includes the use of immunohistochemical (IHC) stains. We used defined histopathologic patterns, histochemical staining, and IHC staining for smooth muscle actin (SMA), desmin, and laminin to characterize suspected non-visceral leiomyosarcoma in dogs at a single academic institution. In a retrospective search, we identified 24 dogs with a definitive or suspected histologic diagnosis of leiomyosarcoma in a non-visceral location. Histopathology results and clinical details were obtained. Biopsy sections were reviewed by a single pathologist using standardized histologic criteria, including light microscopic appearance, immunohistochemistry (more than two-thirds of neoplastic cells labeled with SMA and desmin or laminin), and histochemical staining (minimal-to-mild matrix deposition by Masson trichrome). Of the 24 cases of possible non-visceral leiomyosarcomas, 4 were consistent with a definitive diagnosis of non-visceral leiomyosarcoma (3) or leiomyoma (1) based on the established criteria. Only the leiomyoma had more than two-thirds of neoplastic cells label with all 3 markers; all 3 leiomyosarcomas had more than two-thirds of neoplastic cells label with SMA and laminin. Our data highlight the uncommon nature of non-visceral leiomyosarcoma and the importance of IHC for their diagnosis. A definitive diagnosis could not be made based on SMA alone, and desmin was not useful in this cohort. Further studies are needed to clarify the histopathologic, IHC, and clinical features of canine non-visceral SMA-positive mesenchymal tumors.

Using the canine microbiome to bridge translation of cancer immunotherapy from pre-clinical murine models to human clinical trials.

The microbiome has clearly been established as a cutting-edge field in tumor immunology and immunotherapy. Growing evidence supports the role of the microbiome in immune surveillance, self-tolerance, and response to immune checkpoint inhibitors such as anti PD-L1 and CTLA-4 blockade (1-6). Moreover, recent studies including those using fecal microbial transplantation (FMT) have demonstrated that response to checkpoint immunotherapies may be conferred or eliminated through gut microbiome modulation (7, 8). Consequently, studies evaluating microbiota-host immune and metabolic interactions remain an area of high impact research. While observations in murine models have highlighted the importance of the microbiome in response to therapy, we lack sufficient understanding of the exact mechanisms underlying these interactions. Furthermore, mouse and human gut microbiome composition may be too dissimilar for discovery of all relevant gut microbial biomarkers. Multiple cancers in dogs, including lymphoma, high grade gliomas, melanomas and osteosarcoma (OSA) closely resemble their human analogues, particularly in regard to metastasis, disease recurrence and response to treatment. Importantly, dogs with these spontaneous cancers also have intact immune systems, suggesting that microbiome analyses in these subjects may provide high yield information, especially in the setting of novel immunotherapy regimens which are currently expanding rapidly in canine comparative oncology (9, 10). Additionally, as onco-microbiotic therapies are developed to modify gut microbiomes for maximal responsiveness, large animal models with intact immune systems will be useful for trialing interventions and monitoring adverse events. Together, pre-clinical mechanistic studies and large animal trials can help fully unlock the potential of the microbiome as a diagnostic and therapeutic target in cancer.

Evaluation of the use of a novel bioabsorbable polymer drug-eluting microsphere for transarterial embolization of hepatocellular neoplasia in dogs.

In dogs with non-resectable hepatic neoplasia, treatment options are limited. The objectives of this study were to describe the use of a novel drug-eluting embolic microsphere containing paclitaxel for use during transarterial chemoembolization (TACE), to compare results of liver-specific owner questionnaires and tumor volume pre- and post-TACE, and to measure systemic paclitaxel concentration post-TACE. Client-owned dogs with non-resectable hepatic neoplasia were prospectively enrolled. All owners completed questionnaires validated for the assessment of subjective outcomes in dogs with cancer before the TACE procedure and approximately 4 weeks after the TACE procedure. A CT scan was performed before TACE and 1 month after TACE; results were compared. Blood samples were obtained at specified time points post-TACE to determine systemic paclitaxel concentrations. Seven dogs (median weight: 8.9 kg; range, 4.3-31 kg) were enrolled. TACE was successfully performed in all dogs, and no intra-procedural complications were encountered. Questionnaire scores improved significantly post-TACE. Among the 6 dogs for which full data were available, median pre-TACE tumor volume was 390 cc (range 152-1,484; interquartile range 231-1,139) and median post-TACE tumor volume was 203 cc (range 98-889; interquartile range 151-369), which was significantly (P = .028) lower. All 6 dogs had a reduction in volume at the post-TACE measurement. Mean percent change in tumor volume was -45.6% (95%CI -58.6 to -32.6%). The mean plasma paclitaxel concentration in canine blood peaked at 4 days post-TACE procedure and was 25.7 ng/mL (range = 3.09-110 ng/mL) Median survival time was 629 days (95%CI 18 to upper limit not reached). The use of a novel paclitaxel-eluting microsphere in this cohort of dogs successfully decreased tumor volume significantly after TACE and improved clinical signs. Future investigation into the use of TACE and other similar therapies is warranted due to the promising outcomes noted in this cohort.

Transcriptome Analysis of Tumor-Infiltrating Lymphocytes Identifies NK Cell Gene Signatures Associated With Lymphocyte Infiltration and Survival in Soft Tissue Sarcomas.

Purpose: Clinical successes using current T-cell based immunotherapies have been limited in soft tissue sarcomas (STS), while pre-clinical studies have shown evidence of natural killer (NK) cell activity. Since tumor immune infiltration, especially tumor-infiltrating lymphocytes, is associated with improved survival in most solid tumors, we sought to evaluate the gene expression profile of tumor and blood NK and T cells, as well as tumor cells, with the goal of identifying potential novel immune targets in STS. Experimental Design: Using fluorescence-activated cell sorting, we isolated blood and tumor-infiltrating CD3-CD56+ NK and CD3+ T cells and CD45- viable tumor cells from STS patients undergoing surgery. We then evaluated differential gene expression (DGE) of these purified populations with RNA sequencing analysis. To evaluate survival differences and validate primary DGE results, we also queried The Cancer Genome Atlas (TCGA) database to compare outcomes stratified by bulk gene expression. Results: Sorted intra-tumoral CD3+ T cells showed significant upregulation of established activating (CD137) and inhibitory genes (TIM-3) compared to circulating T cells. In contrast, intra-tumoral NK cells did not exhibit upregulation of canonical cytotoxic genes (IFNG, GZMB), but rather significant DGE in mitogen signaling (DUSP4) and metabolic function (SMPD3, SLC7A5). Tumors with higher NK and T cell infiltration exhibited significantly increased expression of the pro-inflammatory receptor TLR4 in sorted CD45- tumor cells. TCGA analysis revealed that tumors with high TLR4 expression (P = 0.03) and low expression of STMN1 involved in microtubule polymerization (P < 0.001) were associated with significantly improved survival. Conclusions: Unlike T cells, which demonstrate significant DGE consistent with upregulation of both activating and inhibiting receptors in tumor-infiltrating subsets, NK cells appear to have more stable gene expression between blood and tumor subsets, with alterations restricted primarily to metabolic pathways. Increased immune cell infiltration and improved survival were positively correlated with TLR4 expression and inversely correlated with STMN1 expression within tumors, suggesting possible novel therapeutic targets for immunotherapy in STS.