Canine Cancer Research Articles

Synthesis and Antiproliferative Activity of Novel Quercetin-1,2,3-Triazole Hybrids using the 1,3-Dipolar Cycloaddition (Click) Reaction

AbstractTwenty-three new quercetin-1,2,3-triazole hybrids were synthesized in good to quantitative yields via Cu(I)-catalyzed azide-alkyne cycloaddition reaction under microwave irradiation. These new hybrids contain a 1,4-disubstituted 1,2,3-triazole ring at the 3-OH position of quercetin whilst the remaining hydroxyl groups were either protected as methyl or benzyl groups or left unprotected. All the quercetin-1,2,3-triazole hybrids I–IV were evaluated against REM-134 canine mammary cancer cell line, which is used as a translational model for human breast cancer. These new analogues exhibit potent antiproliferative activity against this cancer cell line. Furthermore, the results show that some of the new quercetin-1,2,3-triazole hybrids have better activity than quercetin. Our best inhibitors displayed IC50 values in the range of 41–180 nM, and undoubtedly will have an important impact on the treatment of both canine and human breast cancer.

Comparative oncology analysis using genomic profiling of canine cancers by tissue and liquid biopsy testing reveals actionable somatic alterations with orthologs in human cancers.

e15012 Background: Targeted therapies offer great potential for cancer treatment in humans; however, enrollment in clinical trials may be slow, particularly for rare cancer types. The high homology between human and canine cancer genomes provides an opportunity for comparative oncology analyses that can inform the development of therapeutics for the benefit of both species. Currently, there is no well-established canine cancer genomic database. This study presents preliminary findings from an ongoing initiative aimed at building a foundational database of canine cancer genomic variants and their human orthologs via tissue and liquid biopsy profiling of multiple cancer types. Methods: Matched blood and tissue samples were collected from an all-comers cohort of over 150 cancer-diagnosed, client-owned dogs undergoing surgical resection or tissue sampling by biopsy. Cancer types included those found in both humans and dogs, such as lymphoma, osteosarcoma, mammary gland tumors and melanoma. Blood samples were processed to obtain plasma and white blood cells, and all samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Somatic variants detected by liquid biopsy and tissue testing were mapped to human orthologs targeted by therapeutic agents approved by the FDA or listed in NCCN guidelines, and to orthologs with therapeutic or prognostic levels of evidence annotated in human oncology databases. Results: Somatic alterations were identified in over half of the tissue and/or liquid biopsy samples tested. Results from tissue and liquid biopsy showed high concordance, and liquid biopsy revealed additional alterations that may have been missed in single-site tissue biopsies due to tumor heterogeneity. Of the mutations identified in plasma and/or tissue but absent in matched white blood cells, 10% mapped to a human ortholog listed as a biomarker targeted by either an approved therapeutic agent or a therapeutic currently undergoing clinical trials; examples include NRAS, HRAS and AKT1 mutations. Over 10% of canine cancer patients also harbored orthologs of somatic TP53 mutations reported to be prognostic in multiple human cancers. In particular, TP53 mutations were found in > 20% of canine osteosarcoma patients, which aligns with prior findings in human osteosarcoma and highlights the potential of comparative oncology for cancer types that are rare in humans but common in dogs. Conclusions: The results confirm the homology between canine and human cancers and emphasize the potential to translate genomic knowledge for biomarker discovery and therapeutic development between species. The high concordance between tumor and liquid biopsy findings support the prospect of using a noninvasive blood test to expand the genomic characterization effort to much larger cohorts of cancer-diagnosed dogs.

Comparative Pathobiology of Canine and Human Prostate Cancer: State of the Art and Future Directions.

Simple SummaryProstate cancer (PC) is one of the main types of cancer that affects the male population worldwide. In recent decades, there has been a significant evolution in the methods of diagnosis and treatment, mainly due to the development of new research in the field of molecular biology, allowing for a better understanding of how this cancer develops and progresses from a genetic point of view. In addition to humans, the canine is the only mammal that develops PC spontaneously. However, in dogs, PC has a distinct form, with high lethality rates. This work of scientific characterization of cancer in humans and dogs approaches how differences and similarities already all explain how PC develops and progresses in dogs, with a central tenet of molecular biology, that is, the transport between DNA, RNA and proteins. The understanding of these mechanisms makes it possible to identify new methods of the diagnosis and treatment of the disease, not only in men, but also in dogs.First described in 1817, prostate cancer is considered a complex neoplastic entity, and one of the main causes of death in men in the western world. In dogs, prostatic carcinoma (PC) exhibits undifferentiated morphology with different phenotypes, is hormonally independent of aggressive character, and has high rates of metastasis to different organs. Although in humans, the risk factors for tumor development are known, in dogs, this scenario is still unclear, especially regarding castration. Therefore, with the advent of molecular biology, studies were and are carried out with the aim of identifying the main molecular mechanisms and signaling pathways involved in the carcinogenesis and progression of canine PC, aiming to identify potential biomarkers for diagnosis, prognosis, and targeted treatment. However, there are extensive gaps to be filled, especially when considering the dog as experimental model for the study of this neoplasm in humans. Thus, due to the complexity of the subject, the objective of this review is to present the main pathobiological aspects of canine PC from a comparative point of view to the same neoplasm in the human species, addressing the historical context and current understanding in the scientific field.

Label-free multimodal nonlinear optical imaging of needle biopsy cores for intraoperative cancer diagnosis.

.Significance: Needle biopsy (NB) procedures are important for the initial diagnosis of many types of cancer. However, the possibility of NB specimens being unable to provide diagnostic information, (i.e., non-diagnostic sampling) and the time-consuming histological evaluation process can cause delays in diagnoses that affect patient care.Aim: We aim to demonstrate the advantages of this label-free multimodal nonlinear optical imaging (NLOI) technique as a non-destructive point-of-procedure evaluation method for NB tissue cores, for the visualization and characterization of the tissue microenvironment.Approach: A portable, label-free, multimodal NLOI system combined second-harmonic generation (SHG) and third-harmonic generation and two- and three-photon autofluorescence (2PF, 3PF) microscopy. It was used for intraoperative imaging of fresh NB tissue cores acquired during canine cancer surgeries, which involved liver, lung, and mammary tumors as well as soft-tissue sarcoma; in total, eight canine patients were recruited. An added tissue culture chamber enabled the use of this NLOI system for longitudinal imaging of fresh NB tissue cores taken from an induced rat mammary tumor and healthy mouse livers.Results: The intraoperative NLOI system was used to assess fresh canine NB specimens during veterinary cancer surgeries. Histology-like morphological features were visualized by the combination of four NLOI modalities at the point-of-procedure. The NLOI results provided quantitative information on the tissue microenvironment such as the collagen fiber orientation using Fourier-domain SHG analysis and metabolic profiling by optical redox ratio (ORR) defined by 2PF/(2PF + 3PF). The analyses showed that the canine mammary tumor had more randomly oriented collagen fibers compared to the tumor margin, and hepatocarcinoma had a wider distribution of ORR with a lower mean value compared to the liver fibrosis and the normal-appearing liver. Moreover, the loss of metabolic information during tissue degradation of fresh murine NB specimens was shown by overall intensity decreases in all channels and an increase of mean ORR from 0.94 (standard deviation 0.099) to 0.97 (standard deviation 0.077) during 1-h longitudinal imaging of a rat mammary tumor NB specimen. The tissue response to staurosporine (STS), an apoptotic inducer, from fresh murine liver NB specimens was also observed. The mean ORR decreased from 0.86 to 0.74 in the first 40 min and then increased to 0.8 during the rest of the hour of imaging, compared to the imaging results without the addition of STS, which showed a continuous increase of ORR from 0.72 to 0.75.Conclusions: A label-free, multimodal NLOI platform reveals microstructural and metabolic information of the fresh NB cores during intraoperative cancer imaging. This system has been demonstrated on animal models to show its potential to provide a more comprehensive histological assessment and a better understanding of the unperturbed tumor microenvironment. Considering tissue degradation, or loss of viability upon fixation, this intraoperative NLOI system has the advantage of immediate assessment of freshly excised tissue specimens at the point of procedure.

Establishment of a new cell line of canine inflammatory mammary cancer: IMC-118.

Canine inflammatory mammary cancer (IMC) has long been regarded as an attractive animal model for research into human inflammatory breast cancer (IBC), Although some canine mammary tumour cell lines corresponding to human mammary cancer cell lines have been established, there is still a need to supplement the canine mammary tumour cell bank. The goal of this study was to create a new type of IMC cell line. The primary tumour, IMC-118, was identified as IMC by pathology examination. Immunohistochemistry analysis revealed negative immunoreactivity to oestrogen receptor (ER), but positive immunoreactivity to progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER-2). Immunofluorescence (IF) analysis revealed that the IMC-118 cell line from this primary tumour was negative for ER but positive for PR and HER-2, and was also positive for epithelial and mesenchymal cell markers. This cell line was cultured stably for more than 50 passages and grew well after cryopreservation. In vivo, tumour masses and metastases in the lungs were discovered after inoculating the IMC-118 cells into the nude mice model. As a result, a novel canine IMC cell line, IMC-118, was effectively established, and could be employed as a promising model for immunotherapy and epithelial-mesenchymal transition mechanism of IMC research in both dogs and humans.

Stimulation of Osteoblastic Bone Metastasis by Canine Prostate Cancer

Prostate cancer metastases primarily localize in the bone where they induce a unique osteoblastic response. The mechanisms are not well understood; however, they depend on a complex interplay between prostate cancer cells and the cellular microenvironment in the bone marrow. We hypothesize that prostate cancer cells secreted biological factors that induce new bone formation and either induce or stimulate osteoclastic bone resorption to permit growth of bone metastasis. Dogs are the only other species that develop spontaneous prostate cancer with bone metastases in addition to men. Previously, our laboratory has developed three canine prostate cancer cell lines that mimic human osteoblastic bone metastases when injected into the left cardiac ventricle of nude mice. This study then investigated the effect of three canine prostate cancer cell lines (Ace‐1, Probasco, and LuMa) on bone formation and resorption. Mouse calvaria were treated with conditioned medium (CM) collected from cell lines. A novel calcein uptake assay was developed to measure bone formation and mineralization in vitro. Ace‐1 CM induced predominantly bone resorption in calvaria, while Probasco CM induced marked bone formation, mineralization, and healing of calvaria defects. The expression of osteoblast‐related genes in calvaria showed that Probasco CM stimulated the maturation and differentiation of osteoblasts and inhibited osteoclastogenesis. Both bone modeling and remodeling were involved in Probasco‐induced bone formation and mineralization by inhibiting remodeling with zoledronic acid. RNAseq results from CM treated osteoblasts indicated that the WNT signaling pathway was activated by Probasco compared to either control or Ace‐1. In addition, Calvaria treated with Probasco+hDKK‐1 CM, Probasco transduced with human DKK‐1 cDNA (a WNT antagonist), showed decreased bone formation. In vivo, Our preliminary data showed that DKK‐1 significantly increased the growth of Probasco tumors after intratibial injection of Probasco+hDKK‐1 cells in nude mice, which suggested the autocrine effect of DKK‐1 on tumor cells. In conclusion, Probasco cells induced bone formation and mineralization in vitro that depended on the WNT signaling pathway. Probasco cells serve as a valuable model for studying the mechanisms of osteoblastic bone metastasis in prostate cancer.

Phenotypic and transcriptomic profiles of tumor-conditioned macrophages in dogs, and the translational value of canine cancer immunology research.

Abstract Tumor-associated macrophages (TAMs) are heterogeneous and abundantly present in the tumor stroma. They derive from tissue-resident macrophages and recruited monocytes and play a central role in several aspects of cancer. In murine models, TAMs promote invasion, intravasation, circulatory survival and extravasation of tumor cells. TAMs also promote tumor progression and angiogenesis, remodel the tumor microenvironment, and modulate the adaptive immune system. Evidence suggests TAMs are vital for establishing the premetastatic niche. Historically, family-owned dogs have proven to be reliable cancer models. In contrast to laboratory animals, dogs develop cancer naturally and commonly, co-inhabit our environment, are genetically outbred and immunologically experienced. We hypothesized that canine tumor conditioned monocyte-derived macrophages (TCMs) share many characteristics with M2 macrophages and have a tumor-promoting phenotype as in humans. We collected blood from healthy dogs, isolated peripheral blood monocytes and differentiated them in vitro using tumor conditioned media from three canine cancer lines and M-CSF. Three TCM populations were generated and compared with M1 and M2 macrophages. We assessed the macrophages using multicolor flow cytometry and RNA-sequencing. All TCM showed an increased expression of two M2 markers (CD209a and FcɛRI), while 2/3 TCM populations had increased expression of two additional M2 markers (CD206 and CD11d). Transcriptomic analysis is ongoing. Results so far support the hypothesis that canine TCMs share many similarities with human TCMs, further warranting the use of dogs as cancer immunology models. Supported by grant from Agria Animal Insurances (N-2020-0061) and The Research fund for cancer in dogs (Oslo, Norway)

Characterizing a Canine Urothelial Carcinoma Model of Acquired MEK1/2 Inhibitor Resistance

Activating BRAF mutations are drivers of oncogenesis in several human cancers and in over 70% of canine urothelial carcinomas (cUC). Selective BRAF inhibitors are effective alone and in combination with MEK inhibitors in approximately half of BRAF mutant melanoma cases, but the majority of these patients ultimately develop resistance. Thus, intrinsic and acquired resistance to MAP kinase pathway targeted agents remains a challenge requiring the development of novel therapeutic strategies. Using canine urothelial carcinoma as a spontaneous cancer model to explore treatment of BRAF mutant cancers and acquired drug resistance, we sought to identify determinants of MEK1/2 inhibitor sensitivity and resistance. We applied the human‐derived MAPK Pathway Activity Score (MPAS), a predictor of sensitivity to MEK inhibition in human cancers based on expression levels of 10 downstream targets, to a panel of 32 canine cancer cell lines and found that MPAS correlates with trametinib sensitivity in this canine dataset. MPAS genes with significant (p<0.05) Pearson correlations to sensitivity were CCND1, DUSP6, ETV4, ETV5, and SPRY2. We also identified 30 non‐MPAS genes whose expression levels are significantly correlated with trametinib sensitivity, suggesting their role in de novo sensitivity to trametinib. Each of the 5 urothelial carcinoma lines had high MPAS scores and trametinib IC50 values <10nM. CRISPR‐Cas9 knockouts in two canine urothelial carcinoma lines also identified MEK1 as a critical dependency. To investigate mechanisms of acquired resistance, we generated trametinib‐resistant (TramR) clonal derivatives of the BRAF mutant Tyler1 canine urothelial carcinoma cell line (Tyler1‐TramR), with trametinib IC50 values greater than 500 nM versus 1 nM in parental Tyler1. Tyler1‐TramR cell lines exhibited sustained inhibition of ERK1/2 phosphorylation when treated with trametinib and were also resistant to pharmacologic inhibition of ERK1/2. Collectively, these data suggest an ERK‐independent mechanism of resistance. Gene expression analysis of two Tyler1‐TramR clones identified dramatic downregulation of epithelial markers accompanied by an increase in expression of mesenchymal genes and transcription factors that regulate the epithelial‐to‐mesenchymal transition (EMT). One Tyler1‐TramR clone exhibited metabolic alterations including decreased basal and maximal oxygen consumption rates, diminished spare respiratory capacity, and reduced glycolytic capacity. Overall, the findings in this study suggest that the mechanisms of acquired and intrinsic MEK inhibitor resistance are conserved between man and dog, further elucidating the translational value of cUC as a model to optimize therapies for Ras/Raf/MAP kinase driven cancers.

Review: NF-kB activation in canine cancer.

Spontaneous tumors in dogs share several environmental, epidemiologic, biologic, clinical and molecular features with a wide variety of human cancers, making this companion animal an attractive model. Nuclear factor kappa B (NF-kB) transcription factor overactivation is common in several human cancers, and there is evidence that similar signaling aberrations also occur in canine cancers including lymphoma, leukemia, hemangiosarcoma, mammary cancer, melanoma, glioma, and prostate cancer. This review provides an overview of NF-kB signaling biology, both in health and in cancer development. It also summarizes available evidence of aberrant NF-kB signaling in canine cancer, and reviews antineoplastic compounds that have been shown to inhibit NF-kB activity used in various types of canine cancers. Available data suggest that dogs may be an excellent model for human cancers that have overactivation of NF-kB.

Malignant Alveolar Neoplasm in a 10-Month-Old French Bulldog

Background: Malignant tumors are the main cause of death or euthanasia in animals. The oral cavity ranking fourth in number of occurrences. Epidemiological studies with dogs suggest that canine cancer kills 40-50% of individuals aged over 10 years. In view of the interest of academics and professionals in the healthcare of dogs and cats, this paper reports the case of a 10-month-old bitch, which, despite being a young animal, was affected alveolar rhabdomyosarcoma of abrupt evolution. Case: A 10-month-old French Bulldog bitch, weighing 10 kg, was referred to a veterinary hospital in the city of Rio de Janeiro for care. It had a history of mouth bleeding, after chewing a solid mineral material, edema in the region of the right maxilla, and protusion of the gland of the third eyelid. As the clinical examination also revealed a fracture of the maxillary canine, anti-inflammatory and antibiotics were prescribed, to be administered by the owner once a day for 7 days. During the next clinical examination, carried out one week later, an edema was found in the right region of the mouth, which proved difficult to examine. As the patient had already eaten, an appointment was made for the following day for an intervention int he operating room, where the animal could be anesthetized for better observation of the effected region. Blood was collected for hemogram, urea, creatinine, alkaline phosphatase, ALT, and GGT, and an 8 h food fasting and a 4 h water fasting were recommended. On that date, once the dog had been taken to the operating room, was administered the pre anesthesia, in addition to anesthetic induction and manutention. Upon examining the oral cavity, several loose molars were found on the right side, in addition to a tumoral aspect of the gum; thus, it was decided to collect a small sample of the tumoral mass for histopathology. The surgical specimen was placed in a formalin solution and sent to the laboratory for histopathological processing and diagnosis. One week later, the tumor mass was larger and the edema in the right region of the mouth was much larger than on the day of the procedure. Thus, a computerized tomography was requested to further investigate the alterations that had occurred in such a short time. Due to the results of the histopathology and the CT, an immunohistochemical test was suggested which determined the cell profile and morphology and confirmed the diagnosis of alveolar rhabdomyosarcoma according to clinical suspicion. The animal remained in the veterinary hospital for a further 48 h, during which the clinical condition worsened, with the animal suffering heavy bleeding. As the patient was no longer capable of oral intake of food or water, the decision was made with the consent of the owners to induce a painless death to alleviate the suffering of the animal. However, the owners did not authorize a necropsy. Discussion: Veterinary physicians should be conscious of the treatment of serious illnesses that will not result in a benefit for the patient. They should know when to stop the treatment to not cause further pain and suffering to the animals and their owners. Many of the interventions which aim to treat severe malignant neoplasia will not promote an improvement in quality of life or significantly extend the patient’s survival, and do not justify the suffering they entail. A painless death remains the best alternative in such cases. Keywords: cancer, malignant neoplasm, alveolar rhabdomyosarcoma, oral cavity.

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

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

Feasibility of circulating tumor DNA analysis in dogs with naturally occurring malignant and benign splenic lesions

Comparative studies of naturally occurring canine cancers have provided new insight into many areas of cancer research. Development and validation of circulating tumor DNA (ctDNA) analysis in pet dogs can help address diagnostic needs in veterinary as well as human oncology. Dogs have high incidence of naturally occurring spontaneous cancers, demonstrate molecular heterogeneity and clonal evolution during therapy, allow serial sampling of blood from the same individuals during the course of disease progression, and have relatively compressed intervals for disease progression amenable to longitudinal studies. Here, we present a feasibility study of ctDNA analysis performed in 48 dogs including healthy dogs and dogs with either benign splenic lesions or malignant splenic tumors (hemangiosarcoma) using shallow whole genome sequencing (sWGS) of cell-free DNA. To enable detection and quantification of ctDNA using sWGS, we adapted two informatic approaches and compared their performance for the canine genome. At the time of initial clinical presentation, mean ctDNA fraction in dogs with malignant splenic tumors was 11.2%, significantly higher than dogs with benign lesions (3.2%; p = 0.001). ctDNA fraction was 14.3% and 9.0% in dogs with metastatic and localized disease, respectively (p = 0.227). In dogs treated with surgical resection of malignant tumors, mean ctDNA fraction decreased from 11.0% prior to resection to 7.9% post-resection (p = 0.047 for comparison of paired samples). Our results demonstrate that ctDNA analysis is feasible in dogs with hemangiosarcoma using a cost-effective approach such as sWGS. Additional studies are needed to validate these findings, and determine the role of ctDNA to assess burden of disease and treatment response in dogs with cancer.

Evaluation of the therapeutic potential of masitinib and expression of its specific targets c-Kit, PDGFR-α, PDGFR-β, and Lyn in canine prostate cancer cell lines.

Canine prostate cancer is classified into adenocarcinoma, transitional cell carcinoma with prostatic involvement, and mixed forms. Early metastatic spread leads to poor prognosis and limited treatment options. Masitinib is approved for the treatment of canine mast cell tumours and inhibits tyrosine kinase c-Kit, tyrosine-protein kinase Lyn (Lyn), and platelet-derived growth factor receptors alpha and beta (PDGFR-α, PDGFR-β), which are known to be expressed in canine prostate cancer. The aim of this study was to evaluate masitinib in an in vitro model consisting of cell lines from primary prostate adenocarcinoma, the associated lymph node metastasis of the same patient, and transitional cell carcinoma. To assess the suitability of the model system, the targets of masitinib were investigated by immunocytochemistry in the cell lines and by immunohistochemistry in the respective formalin-fixed, paraffin-embedded (FFPE) original neoplastic tissue. After exposure to masitinib, cell viability, cell count, apoptosis induction, and protein expression of c-Kit, Lyn, PDGFR-α, and PDGFR-β were assessed. To hedge the efficacy, two application protocols of masitinib (single application or 12-h double-dose regimen) were compared. Immunocytochemical and immunohistochemical analysis revealed increased Lyn, PDGFR-α, and PDGFR-β expression in cell lines and FFPE original neoplastic tissue compared to healthy prostate tissue. Masitinib exposure increased apoptosis, while the cell counts and cell viability decreased in a dose- and application interval-dependent manner, with increased impact in the 12-h double-dose regimen. These in vitro effects of masitinib in canine prostate cancer and associated metastasis support further in vivo research and modifications of the clinical treatment protocol in future studies.

Developing a translational murine-to-canine pathway for an IL-2/agonist anti-CD40 antibody cancer immunotherapy.

Human and canine sarcomas are difficult to treat soft tissue malignancies with an urgent need for new improved therapeutic options. Local recurrence rates for humans are between 10%–30%, and 30%–40% develop metastases. Outcomes for dogs with sarcoma vary with grade but can be similar. Pet dogs share the human environment and represent human cancer with genetic variation in hosts and tumours. We asked if our murine studies using genetically identical mice and cloned tumour cells were translatable to larger, genetically diverse domestic dogs with naturally occurring tumours, to (i) develop a canine cancer therapeutic, and (ii) to use as a translational pathway to humans. Our murine studies showed that intra‐tumoral delivery of interleukin‐2 (IL‐2) plus an agonist anti‐CD40 antibody (Ab) induces long‐term curative responses ranging from 30% to 100%, depending on tumour type. We developed an agonist anti‐canine‐CD40 Ab and conducted a phase I dose finding/toxicology 3 + 3 clinical trial in dogs (n = 27) with soft tissue sarcomas on account of suitability for intratumoral injection and straightforward monitoring. Dogs were treated with IL‐2 plus anti‐CD40 antibody for 2 weeks. Three dose levels induced tumour regression with minimal side effects, measured by monitoring, haematological and biochemical assays. Importantly, our mouse and canine studies provide encouraging fundamental proof‐of‐concept data upon which we can develop veterinary and human immunotherapeutic strategies.

Non-Exosomal and Exosome-Derived miRNAs as Promising Biomarkers in Canine Mammary Cancer.

Canine mammary cancer (CMC), similar to human breast cancer (HBC) in many aspects, is the most common neoplasm associated with significant mortality in female dogs. Due to the limited therapy options, biomarkers are highly desirable for early clinical diagnosis or cancer progression monitoring. Since the discovery of microRNAs (miRNAs or miRs) as post-transcriptional gene regulators, they have become attractive biomarkers in oncological research. Except for intracellular miRNAs and cell-free miRNAs, exosome-derived miRNAs (exomiRs) have drawn much attention in recent years as biomarkers for cancer detection. Analysis of exosomes represents a non-invasive, pain-free, time- and money-saving alternative to conventional tissue biopsy. The purpose of this review is to provide a summary of miRNAs that come from non-exosomal sources (canine mammary tumor, mammary tumor cell lines or canine blood serum) and from exosomes as promising biomarkers of CMC based on the current literature. As is discussed, some of the miRNAs postulated as diagnostic or prognostic biomarkers in CMC were also altered in HBC (such as miR-21, miR-29b, miR-141, miR-429, miR-200c, miR-497, miR-210, miR-96, miR-18a, miR19b, miR-20b, miR-93, miR-101, miR-105a, miR-130a, miR-200c, miR-340, miR-486), which may be considered as potential disease-specific biomarkers in both CMC and HBC.

E-cadherin Expression in Canine Gastric Carcinomas: Association with Clinicopathological Parameters.

E-cadherin (E-cad) is a cell-adhesion molecule known for its tumor-invasion suppressor function. E-cad expression was examined immunohistochemically in a series of canine tissue samples, including normal gastric mucosa (NGM; n = 3), gastric carcinomas (GC; n = 33), adjacent non-neoplastic mucosa (NNM; n = 32), neoplastic emboli (n = 16) and metastatic lesions (n = 9). The relationship between E-cad expression and clinicopathological features were investigated. In NGM, epithelial cells showed strong latero-lateral membranous expression of E-cad, and this pattern was considered normal. The membranous staining was preserved in all specimens of NNM (100%), whereas abnormal E-cad expression was found in 87.9% of the GCs. A marked difference in E-cad expression was observed between normal and malignant tissues (p < 0.0002). Abnormal E-cad expression was significantly more frequent in poorly/undifferentiated carcinomas (96%) and diffuse (95%) and indeterminate carcinomas (100%) than in well-differentiated/intestinal ones (62.5%; p = 0.0115 and p = 0.0392, respectively). There was significant association between abnormal E-cad expression and the depth of invasion (p = 0.0117), and the presence neoplastic emboli (p = 0.0194). No statistically significant differences in E-cad expression were observed concerning tumor location, histological type according to WHO classification, and presence of metastatic lesions. Therefore, deregulation of E-cad expression may play a role in canine gastric carcinogenesis and in tumor progression; moreover, it might be a prognostic tool for canine gastric cancer.

Accurate Three-Dimensional Thermal Dosimetry and Assessment of Physiologic Response Are Essential for Optimizing Thermoradiotherapy.

Simple SummaryMany clinical trials have shown benefit for adding hyperthermia (heat) treatment to radiotherapy. Despite overall success, some patients do not derive maximum benefit from this combination treatment. Tumor hypoxia (low oxygen concentration) is a major cause for radiotherapy treatment resistance. In this paper, we examine the question of whether hyperthermia reduces hypoxia and, if so, whether reduction in hypoxia is associated with treatment outcome. The review is focused mainly on several clinical trials conducted in humans and companion dogs with cancer treated with hyperthermia and radiotherapy. Detailed measurements of temperature, hypoxia and perfusion were made and compared with treatment outcome. These analyses show that reoxygenation after hyperthermia occurs in patients and is related to treatment outcome. Further, reoxygenation is most likely caused by variable intra-tumoral temperatures that improve perfusion and reduce oxygen consumption rate. Directions for future research on this important issue are indicated.Numerous randomized trials have revealed that hyperthermia (HT) + radiotherapy or chemotherapy improves local tumor control, progression free and overall survival vs. radiotherapy or chemotherapy alone. Despite these successes, however, some individuals fail combination therapy; not every patient will obtain maximal benefit from HT. There are many potential reasons for failure. In this paper, we focus on how HT influences tumor hypoxia, since hypoxia negatively influences radiotherapy and chemotherapy response as well as immune surveillance. Pre-clinically, it is well established that reoxygenation of tumors in response to HT is related to the time and temperature of exposure. In most pre-clinical studies, reoxygenation occurs only during or shortly after a HT treatment. If this were the case clinically, then it would be challenging to take advantage of HT induced reoxygenation. An important question, therefore, is whether HT induced reoxygenation occurs in the clinic that is of radiobiological significance. In this review, we will discuss the influence of thermal history on reoxygenation in both human and canine cancers treated with thermoradiotherapy. Results of several clinical series show that reoxygenation is observed and persists for 24–48 h after HT. Further, reoxygenation is associated with treatment outcome in thermoradiotherapy trials as assessed by: (1) a doubling of pathologic complete response (pCR) in human soft tissue sarcomas, (2) a 14 mmHg increase in pO2 of locally advanced breast cancers achieving a clinical response vs. a 9 mmHg decrease in pO2 of locally advanced breast cancers that did not respond and (3) a significant correlation between extent of reoxygenation (as assessed by pO2 probes and hypoxia marker drug immunohistochemistry) and duration of local tumor control in canine soft tissue sarcomas. The persistence of reoxygenation out to 24–48 h post HT is distinctly different from most reported rodent studies. In these clinical series, comparison of thermal data with physiologic response shows that within the same tumor, temperatures at the higher end of the temperature distribution likely kill cells, resulting in reduced oxygen consumption rate, while lower temperatures in the same tumor improve perfusion. However, reoxygenation does not occur in all subjects, leading to significant uncertainty about the thermal–physiologic relationship. This uncertainty stems from limited knowledge about the spatiotemporal characteristics of temperature and physiologic response. We conclude with recommendations for future research with emphasis on retrieving co-registered thermal and physiologic data before and after HT in order to begin to unravel complex thermophysiologic interactions that appear to occur with thermoradiotherapy.

Canine and murine models of osteosarcoma.

Osteosarcoma (OS) is the most common malignant bone tumor in children. Despite efforts to develop and implement new therapies, patient outcomes have not measurably improved since the 1980s. Metastasis continues to be the main source of patient mortality, with 30% of cases developing metastatic disease within 5 years of diagnosis. Research models are critical in the advancement of cancer research and include a variety of species. For example, xenograft and patient-derived xenograft (PDX) mouse models provide opportunities to study human tumor cells in vivo while transgenic models have offered significant insight into the molecular mechanisms underlying OS development. A growing recognition of naturally occurring cancers in companion species has led to new insights into how veterinary patients can contribute to studies of cancer biology and drug development. The study of canine cases, including the use of diagnostic tissue archives and clinical trials, offers a potential mechanism to further canine and human cancer research. Advancement in the field of OS research requires continued development and appropriate use of animal models. In this review, animal models of OS are described with a focus on the mouse and tumor-bearing pet dog as parallel and complementary models of human OS.

Vet-ICD-O-Canine-1, a System for Coding Canine Neoplasms Based on the Human ICD-O-3.2.

Simple SummaryThe development of a widely accepted and comparable animal cancer registration system lacks standardized animal cancer coding. The GIVCS group have developed a comparative coding system for canine neoplasms—Vet-ICD-O-canine-1—compatible with the human ICD-O-3.2 and consistent with the currently recognized classification schemes for canine tumors. This system comprises 335 topography codes and 534 morphology codes allowing the collection of consistent epidemiologic canine cancer data and offering a robust framework for comparative oncology studies.Cancer registries are fundamental tools for collecting epidemiological cancer data and developing cancer prevention and control strategies. While cancer registration is common in the human medical field, many attempts to develop animal cancer registries have been launched over time, but most have been discontinued. A pivotal aspect of cancer registration is the availability of cancer coding systems, as provided by the International Classification of Diseases for Oncology (ICD-O). Within the Global Initiative for Veterinary Cancer Surveillance (GIVCS), established to foster and coordinate animal cancer registration worldwide, a group of veterinary pathologists and epidemiologists developed a comparative coding system for canine neoplasms. Vet-ICD-O-canine-1 is compatible with the human ICD-O-3.2 and is consistent with the currently recognized classification schemes for canine tumors. It comprises 335 topography codes and 534 morphology codes. The same code as in ICD-O-3.2 was used for the majority of canine tumors showing a high level of similarity to their human counterparts (n = 408). De novo codes (n = 152) were created for specific canine tumor entities (n = 126) and topographic sites (n = 26). The Vet-ICD-O-canine-1 coding system represents a user-friendly, easily accessible, and comprehensive resource for developing a canine cancer registration system that will enable studies within the One Health space.

Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer

BackgroundInflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This...