Thyroid tumors represent 1-3% of canine cancers, with most tumors classified as follicular carcinomas and less frequently as medullary carcinomas. In comparison, only 10-15% of human thyroid cancers are follicular and 2% are medullary, with prevalent activating mutations in BRAF and NRAS, or RET, respectively. A cohort of canine thyroid carcinomas underwent histopathological (n=60 and paired molecular (n=30, WES and/or RNAseq) exploration. Clustering of tumor transcriptomes produced 2 groups; T1 and T2 clusters comprised of follicular thyroid carcinomas (FTC) and medullary thyroid carcinomas (MTC), respectively. Tumors were histologically typed as follicular, compact, and follicular-compact on blinded review, with most MTC classified as compact with rare follicular-compact appearance, while FTC displayed all 3 patterns. FTC samples had significantly elevated levels of ERBB2 and HER2 protein, and RET signaling was up-regulated in MTC. Recurrent somatic mutations in DNMT1, STAT2, SALL4, HSP90AA1, MEN1, MUC4, THRAP3, CDK4, NOTCH2, and THRAP3 were identified in at least 10% of samples. Individual variants were also identified in KRAS, ARAF, GNAS, and ERBB2. Additionally, we identified fusion genes that included TG, FGFR2, and PAX8. These data suggest that canine MTC, like their human counterparts, may be driven by RET signaling. In contrast, FTC show limited reliance on RAS/RAF signaling, prevalent in human TC, for oncogenic progression. Across the analyzed samples, 60% of tumors had mutations in at least one DNA repair-related pathway, suggesting that the accumulation of DNA damage may drive cancer progression in canine thyroid tumors. Elevated HER2 protein staining was associated with shorter progression free survival (PFS). Thyroid tumor size (>4.25 cm) was also associated with shorter overall survival and PFS, consistent with previous reports; however, metastasis at diagnosis was not correlated with outcome. Additional studies are warranted to explore the utility of these biomarkers to improve diagnosis and treatment of thyroid carcinoma in dogs. ### Competing Interest Statement The authors have declared no competing interest.

Thyroid tumors represent 1-4% of cancers in both dogs and humans. Most canine tumors are follicular (FTC) or medullary carcinomas (MTC), unlike humans, where only 10-15% are FTC and 2% are MTC, with BRAF/NRAS or RET mutations, respectively. Here, we conduct histological and molecular analyses of canine thyroid tumors. Transcriptionally, elevated ERBB2 expression characterizes FTC tumors, whereas MTC tumors show upregulated RET signaling. Elevated HER2 protein-staining and larger tumor size associate with shorter progression-free survival. Recurrent mutations are rarely observed with potential driver variants in MEN1 (10%), KRAS (7%), and TSHR (3%), among others. Notably, mutations in DNA repair pathway genes are the most consistently shared across tumors, occurring in 60% of cases. Thus, the genomic profile of canine FTC differs significantly from that of humans, with limited reliance on RAS/RAF signaling for oncogenic progression. Conversely, RET signaling likely underlies tumorigenesis in both canine and human MTC.

Supplementary Table 1 contains the inclusion and exclusion criteria for pet dogs considered for enrollment into the SOC and SOC + S clinical trial arms.

AbstractCanine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features. Histologically, canine STS sub-types are difficult to discern at the light microscopic level due to their overlapping features. Thus, genomic, and transcriptomic profiling of canine STS may prove valuable in differentiating the diverse sub-types of mesenchymal neoplasms within this group. To this purpose we sought to characterize the transcript expression and genomic mutation profiles of canine STS. To delineate transcriptomic sub-types, hierarchical clustering was used to identify 4 groups with district expression profiles. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B (PDGFB) and collagen genes. Sensitivity to imatinib was evaluated in a canine STS cell line also bearing aPDGFBfusion. Using whole exome sequencing, recurrent driver variants were identified in the cancer genesKMT2D(21% of the samples) andTP53(21%) along with copy number losses of RB1 and CDKN2A. Gene amplifications and resulting transcript increases were identified in genes on chromosomes 13, 14, and 36. A subset of STS was identified with high T-cell infiltration. This multi-omics approach has defined canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for therapy.

Supplementary Table 4 contains a summary of the outcomes, with statistical comparisons, of the dogs enrolled in the SOC + sirolimus clinical trial arm. This includes the DFI and outcomes of the groups of dogs stratified by tumor location and ALP status.

ObjectiveThe aim of this study was to describe the therapeutic outcomes of dogs with locally advanced salivary gland carcinomas (SGC) following stereotactic body radiation therapy (SBRT).MethodsA single institution retrospective study was conducted of client-owned dogs with macroscopic SGC treated with SBRT. Patient signalment, clinical characteristics, and treatment parameters were recorded. Clinical benefit was determined based on follow-up physical examination and medical history. Progression-free interval (PFI), median survival time (MST), and disease-specific survival (DSS) were calculated using Kaplan–Meier analysis. Acute and late toxicity were recorded according to Veterinary Radiation Therapy Oncology Group (VRTOG) criteria.ResultsSix patients were included in the study. Tumor origins were mandibular (n = 3), parotid (n = 2), and zygomatic (n = 1) salivary glands. The SBRT prescription was 10 Gy × 3 daily or every other day. All patients (100%) experienced clinical benefit from treatment at a median time of 34 days (range 28–214). No local or regional nodal failure was reported following SBRT. Progressive pulmonary metastatic disease was documented in three dogs (50%). The median PFI was 260 days (range 43–1,014) and the MST was 397 days (range 185–1,014). Median DSS was 636 days (range 185–1,014). Four dogs (66.6%) died of confirmed or suspected metastatic SGC. The reported acute side effects included grade 2 mucositis (n = 1) and vision loss (n = 1). No late side effects were recorded.ConclusionThis study suggests that SBRT may provide durable local control for invasive SGC in dogs. Further investigation in a larger cohort of patients is warranted. The incidence of reported acute and late toxicity was low.

Supplementary Table 2 contains the summary comparison of both the Intent-to-treat and Per-protocol analyses of clinical outcomes for dogs enrolled in Standard of Care (SOC) and Standard of Care + sirolimus

Soft tissue sarcomas (STS) are a heterogenous group of mesenchymal tumors representing over 50 distinct types with overlapping histological features and non-specific anatomical locations. Currently, localized sarcomas are treated with surgery + / − radiation in both humans and dogs with few molecularly targeted therapeutic options. However, to improve precision-based cancer therapy through trials in pet dogs with naturally occurring STS tumors, knowledge of genomic profiling and molecular drivers in both species is essential. To this purpose, we sought to characterize the transcriptomic and genomic mutation profiles of canine STS subtypes (fibrosarcoma, undifferentiated pleomorphic sarcoma, and peripheral nerve sheath tumors), by leveraging RNAseq, whole exome sequencing, immunohistochemistry, and drug assays. The most common driver mutations were in cell cycle/DNA repair (31%, TP53 -21%) and chromatin organization/binding (41%, KMT2D -21%) genes. Similar to a subset of human sarcomas, we identified fusion transcripts of platelet derived growth factor B and collagen genes that predict sensitivity to PDGFR inhibitors. Transcriptomic profiling grouped these canine STS tumors into 4 clusters, one PNST group (H1), and 3 FSA groups selectively enriched for extracellular matrix interactions and PDFGB fusions (H2), homeobox transcription factors (H3), and elevated T-cell infiltration (H4). This multi-omics approach provides insights into canine STS sub-types at a molecular level for comparison to their human counterparts, to improve diagnosis, and may provide additional targets for chemo- and immuno-therapy.

Supplementary Fig. 1 depicts the chronology of clinical trial visits and associated procedures for dogs enrolled in this clinical trial.

Supplementary Fig. 2 shows the walk-in pharmacokinetics, both measured results and simulated values, for dogs treated with oral sirolimus in preparation for the definitive adjuvant trial.

T-cell leukemia/lymphoma accounts for roughly 30% of all types of lymphoproliferative neoplasia in dogs. Two forms of T-cell lymphoma (T-zone and peripheral T-cell lymphoma) exhibit breed-specific predilections. During the course of routine immunophenotyping, we observed a breed-specific presentation of a unique form of T-cell leukaemia in young English bulldogs. To describe the clinical presentation and outcome of a novel T-cell leukaemia in English bulldogs and determine the frequency of this neoplasm in other breeds. The Clinical Hematopathology database, containing immunophenotyping data from peripheral blood of nearly 11 900 dogs, was queried for the phenotype observed in young English bulldogs: CD45(+)CD4(-)CD8(-)CD5(+)CD3(+) class II major histocompatibility complex (MHC)-low T-cell leukaemia. Clinical presentation, treatment, and survival data were collected for a subset of cases. Fifty-five English bulldog cases and 64 cases of other breeds were identified. No other breed was represented by >5 cases. Complete medical records were obtained for 50 bulldogs. Median age at diagnosis was 3 years and 76% of cases were male. Median lymphocyte count was 44 286 lymphocytes/mu l (range, 1800-317 684/mu l) and lymphocytes were described as small to intermediate-sized. Many dogs were thrombocytopenic and had liver and spleen involvement, but not lymphadenopathy. Bulldogs that received multi-agent chemotherapy had longer median survival times (83 days) compared to dogs that received no treatment (6 days) or less aggressive therapy (15 days) (p = .001). Non-bulldogs had similar outcomes. CD4(-)CD8(-) class II MHC-low T-cell leukaemia has an aggressive clinical course and predilection for young English bulldogs. Breed-specific presentation suggests an underlying genetic cause.

Canine soft tissue sarcomas (STS) are a heterogenous group of malignant tumors arising from mesenchymal cells of soft tissues. This simplified collective of tumors most commonly arise from subcutaneous tissues, are treated similar clinically, and conventionally exclude other sarcomas with more definitive anatomical, histological, or biological features including hemangiosarcoma, lymphangiosarcoma, histiocytic sarcoma, synovial sarcoma, leiomyosarcoma, and rhabdomyosarcoma. Histologically, canine STS sub‐types are difficult to discern at the light microscopic level due to their overlapping microscopic features. Thus, genomic, and transcriptomic profiling of canine STS may prove more valuable in differentiating the diverse sub‐types of mesenchymal neoplasms within this grouping. To this purpose we sought to characterize the mutation and expression profiles of canine STS. In this study we used whole exome capture (Agilent Sure Select Canine V2) to sequence 29 tumors, grade I‐III, from non‐visceral locations, and matched normal samples, along with RNAseq of tumor samples. For the WES data, 40‐170 million 150 bp paired‐end reads were obtained from Illumina sequencing which were mapped against CanFam3.1 genome via BWA and short variants were called and annotated Mutect2 and VEP tools, respectively. The median depth of sequencing for normals and tumors were 97X (range: 35X–128X) and 110X (range: 22X–133X), respectively. Additionally, RNAseq reads were sequenced from 29 tumors and mapped against CanFam3.1 genome via STAR and gene count data was generated by HTSeq‐count tool. The total number of somatic variants identified across 29 samples ranged from 275 to 4,196, of which, 6% to 31% were annotated as cDNA variants. The protein coding mutations per callable megabase ranged from 0.3 to 37.3. Known cancer genes (COSMIC database) with recurrent mutations were KMT2D(21% of the samples), TP53 (21%), CNTNAP2(14%) genes. Using the RNAseq data, we identified three samples carrying driver fusions of platelet derived growth factor B (PDGFB) and collagen genes. These gene fusions were confirmed through Sanger sequencing of amplified cDNA. To delineate the transcriptomic sub‐types, we used hierarchical and K‐means clustering methods. This resulted in 4 groups with similar expression profiles comprising of 11, 4, 8 and 6 samples in each. Further, genes set variation analysis (GSVA) was used to profile the distinct pathways across all 4 clusters. Preliminary data indicates that the 11‐sample and 6‐sample clusters had up‐regulated non‐homologous end joining pathway and immune response pathways, respectively. Further study of genes and pathways that are enriched in sample clusters will help in categorizing the canine STS tumors for diagnosis and treatment.

A 4-year-old castrated male golden retriever dog was brought to a veterinary teaching hospital for evaluation of acute progressive paraparesis. Neurological examination indicated a spinal cord lesion between the third thoracic vertebra and third lumbar vertebrae. Magnetic resonance imaging (MRI) revealed an intradural, extra medullary, and/or intramedullary mass centered over the eleventh and twelfth thoracic disc space. The dog underwent cytoreductive surgery and histopathologic analysis diagnosed a nephroblastoma. Following this, the dog underwent multimodal therapy, including multiple surgeries, 2 courses of radiation, and combination chemotherapy. The dog had serial restaging using MRI, computed tomography (CT), and fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography throughout the course of therapy. The dog survived 350 d from date of first presentation until humane euthanasia was elected due to worsening of neurologic status. During postmortem examination, extensive infiltration of the spinal cord by nephroblastoma cells was discovered as well as pulmonary metastatic disease. Key clinical message: Based on the literature search, this is the first case in which surgery, radiation therapy, and chemotherapy were all used for the treatment of canine spinal nephroblastoma. This case report details the aggressive nature of a case of canine spinal nephroblastoma despite multi-modal therapy.

The purpose of this bi-institutional retrospective study was to determine whether, in dogs treated with limb amputation and adjunctive chemotherapy for osteosarcoma, oncologic outcomes are impacted by either: (1) baseline cancer pain severity, or (2) the approaches used for perioperative pain management. Data were extracted from the medical records of 284 dogs that underwent both limb amputation and chemotherapy (carboplatin and/or doxorubicin) between 1997 and 2017 for localized (non-metastatic) osteosarcoma of the appendicular skeleton. Kaplan-Meier survival curves and Cox proportional hazard (PH) models were used to determine the impact that retrospectively scored baseline pain levels (high vs. low) and various analgesic and local anaesthetic treatments had on both metastasis-free survival and all-cause mortality. For the entire population, the median disease free interval and median overall survival times were 253 and 284 days, respectively. Baseline pain was rated as "low" in 84 dogs, and "high" in 190 dogs; pain severity had no detectable effect on either metastasis-free survival or all-cause mortality. When accounting for the potential influences of known prognostic factors, dogs treated with what was characterized as a high-intensity perioperative analgesic plan (including both a non-steroidal anti-inflammatory drug [NSAID] and a bupivacaine-eluting soaker catheter placed at the amputation site) had a higher probability of survival than dogs treated with a low-intensity perioperative analgesic plan (neither an NSAID, nor a soaker catheter); the median overall survival times were 252 and 378 days, respectively (hazard ratio: 2.922; p = .020).

Abstract Background The safety and efficacy of stereotactic body radiation therapy (SBRT) in the treatment of localized nasal lymphoma in cats has not been described. Hypothesis Stereotactic body radiation therapy with or without adjuvant chemotherapy is an effective and well‐tolerated treatment for localized nasal lymphoma in cats. Animals Thirty‐two client owned cats referred to Colorado State University for the treatment of nasal lymphoma. Methods Retrospective study of cats treated with SBRT between 2010 and 2020 at Colorado State University. Diagnosis of nasal lymphoma was obtained via cytology or histopathology. Signalment, radiation protocol, concurrent treatments, adverse effects, and survival were recorded. Results Progression free survival was 225 days (95% CI 98–514) and median survival time (MST) was 365 days (95% CI 123–531). No significant difference in survival was identified between cats that received 1 versus greater than 1 fraction (MST 427 vs. 123 days, P = 0.88). Negative prognostic factors included cribriform lysis (MST 121 vs. 876 days, P = 0.0009) and intracalvarial involvement (MST 100 vs. 438 days, P = 0.0007). Disease progression was noted in 38% (12/32), locally in 22% (7/32), and systemically in 16% (5/32). No cats developed acute adverse effects. Ten cats developed late adverse effects: keratitis/keratitis sicca (n = 2), alopecia (n = 4), and leukotrichia (n = 4). Twenty‐four cats (75%) had signs consistent with chronic rhinitis. Conclusions SBRT is effective and well tolerated for treating localized nasal lymphoma in cats. Outcomes for cats with lower stage disease (canine modified Adam's stage 3 and lower) are comparable to historic data of cats treated with fractionated radiation therapy.

Transitional cell carcinoma (TCC), also known as urothelial carcinoma, is the most common bladder cancer in humans and dogs. Approximately one-quarter of human TCCs are muscle-invasive and associated with a high risk of death from metastasis. Canine TCC (cTCC) tumours are typically high-grade and muscle-invasive. Shared similarities in risk factors, histopathology, and clinical presentation suggest that cTCC may serve as a model for the assessment of novel therapeutics that may inform therapies for human muscle-invasive TCC. The goal of this study was to characterize cTCC at the molecular level to identify drivers of oncogenesis and druggable targets. We performed whole exome sequencing (WES) of 11 cTCC tumours and three matched normal samples, identifying 583 variants in protein-coding genes. The most common variant was a V-to-E missense mutation in BRAF, identified in 4 out of 11 samples (36%) via WES. Sanger sequencing identified BRAF variants in 8 out of the same 11 cTCC samples, as well as in 22 out of 32 formalin-fixed paraffin embedded (FFPE) cTCC samples, suggesting an overall prevalence of 70%. RNA-Seq was performed to compare the gene expression profiles of cTCC tumours to normal bladder tissue. cTCC tumours exhibited up-regulation of genes involved in the cell cycle, DNA repair, and antiviral immunity. We also analysed the immune landscape of cTCC using immune gene signatures and immunohistochemical analysis. A subset of tumours had characteristics of a hot tumour microenvironment and exhibited high expression of signatures associated with complete response to PD-1/PD-L1 blockade in human bladder cancer.

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.

Osteosarcoma affects about 2.8% of dogs with cancer, with a one-year survival rate of approximately 45%. The purpose of this study was to characterize the mutation and expression profiles of canine osteosarcoma associated with outcome in samples from dogs treated by amputation and chemotherapy. The number of somatic variants identified across 26 samples ranged from 145 to 2,697 with top recurrent mutations observed in TP53 (82% of the samples) and SETD2 (22%). Additionally, 47 cancer genes were identified with copy number variations in at least 58% of the samples. We observed transcriptional down-regulation of myogenesis genes and up-regulation of extracellular matrix genes in tumors compared to normal bone. Patients with longer disease-free intervals (DFI) showed increased transcript levels of anti-tumor immune response genes. Wild-type/NULL TP53 mutation status and high pre-treatment blood monocyte counts were associated with a shorter DFI. Immune cell infiltration was quantified via immunohistochemistry and gene expression profiling. CD3+ and MAC387+ myeloid cell quantifications were not significantly associated with outcome. Expression of immune related genes PDL-1, CD160 and ICOS were correlated with T-cell abundance. Overall, the association of gene expression and mutation profiles to outcome provides insights into pathogenesis and therapeutic interventions in osteosarcoma patients.

Osteosarcoma affects about 2.8% of dogs with cancer, with a one-year survival rate of approximately 45%. The purpose of this study was to characterize mutation and expression profiles of osteosarcoma and its association with outcome in dogs. The number of somatic variants identified across 26 samples ranged from 145 to 2,697 with top recurrent mutations observed in TP53 and SETD2 . Additionally, 47 cancer genes were identified with copy number variations. Missense TP53 mutation status and low pre-treatment blood monocyte counts were associated with a longer disease-free interval (DFI). Patients with longer DFI also showed increased transcript levels of anti-tumor immune response genes. Although, T-cell and myeloid cell quantifications were not significantly associated with outcome; immune related genes, PDL-1 and CD160 , were correlated with T-cell abundance. Overall, the association of gene expression and mutation profiles to outcome provides insights into pathogenesis and therapeutic interventions in osteosarcoma patients.

Histiocytic sarcoma is an aggressive hematopoietic malignancy of mature tissue histiocytes with a poorly understood etiology in humans. A histologically and clinically similar counterpart affects flat-coated retrievers (FCRs) at unusually high frequency, with 20% developing the lethal disease. The similar clinical presentation combined with the closed population structure of dogs, leading to high genetic homogeneity, makes dogs an excellent model for genetic studies of cancer susceptibility. To determine the genetic risk factors underlying histiocytic sarcoma in FCRs, we conducted multiple genome-wide association studies (GWASs), identifying two loci that confer significant risk on canine chromosomes (CFA) 5 ( P wald = 4.83x10 -9 ) and 19 ( P wald = 2.25x10 -7 ). We subsequently undertook a multi-omics approach that has been largely unexplored in the canine model to interrogate these regions, generating whole genome, transcriptome, and chromatin immunoprecipitation sequencing. These data highlight the PI3K pathway gene PIK3R6 on CFA5, and proximal candidate regulatory variants that are strongly associated with histiocytic sarcoma and predicted to impact transcription factor binding. The CFA5 association colocalizes with susceptibility loci for two hematopoietic malignancies, hemangiosarcoma and B-cell lymphoma, in the closely related golden retriever breed, revealing the risk contribution this single locus makes to multiple hematological cancers. By comparison, the CFA19 locus is unique to the FCR and harbors risk alleles associated with upregulation of TNFAIP6 , which itself affects cell migration and metastasis. Together, these loci explain ~35% of disease risk, an exceptionally high value that demonstrates the advantages of domestic dogs for complex trait mapping and genetic studies of cancer susceptibility.