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Feline exocrine pancreatic carcinoma has been reported to be an aggressive tumor with a high metastatic rate and poor prognosis. Studies reporting long-term outcome of cats after surgical removal of solitary pancreatic carcinomas are rare, due to the uncommon diagnosis and paucity of cats who undergo treatment. In this study, nine cases of feline exocrine pancreatic carcinoma from seven academic and private practice veterinary hospitals were reviewed to examine the outcome in cats undergoing surgical removal of the mass. The median postsurgical survival time for the nine cats was 316.5 days (range, 25–964 days), with three cats alive at a median follow-up time of 309 days. This study demonstrates that surgical removal of pancreatic exocrine tumors in cats with localized disease can result in survival times of over 300 days.

The role of comparative oncology in translational research is receiving increasing attention from drug developers and the greater biomedical research community. Pet dogs with spontaneous cancer are important and underutilized translational models, owing to dogs’ large size and relative outbreeding, combined with their high incidence of certain tumor histotypes with significant biological, genetic, and histological similarities to their human tumor counterparts. Dogs with spontaneous tumors naturally develop therapy resistance and spontaneous metastasis, all in the context of an intact immune system. These fundamental features of cancer biology are often lacking in induced or genetically engineered preclinical tumor models and likely contribute to their poor predictive value and the associated overall high failure rate in oncology drug development. Thus, the conduct of clinical trials in pet dogs with naturally occurring cancer represents a viable surrogate and valuable intermediary step that should be increasingly incorporated into the cancer drug discovery and development pipeline. The development of molecular-targeted therapies has resulted in an expanded role of the pathologist in human oncology trials, and similarly the expertise of veterinary pathologists will be increasingly valuable to all phases of comparative oncology trial design and conduct. In this review, we provide a framework of clinical, ethical, and pathology-focused considerations for the increasing integration of translational research investigations in dogs with spontaneous cancer as a means to accelerate clinical cancer discovery and drug development.

Background KIT inhibitors, such as toceranib (TOC), and vinblastine (VBL) have not been prospectively compared in the treatment of macroscopic mast cell tumors (MCTs). Also, it is unknown whether VBL or TOC is superior for treating MCT without c‐kit mutations. Hypothesis/Objectives To determine the value of KIT genotyping and localization in treatment decisions for dogs with macroscopic MCT. We hypothesized that c‐kit mutated MCT would have a better response to TOC than VBL. Animals Eighty‐eight client‐owned dogs with macroscopic MCT. Methods Prospective, randomized trial. Dogs were randomized to TOC (2.75 mg/kg EOD) or VBL (2.5 mg/m2 weekly × 4 then EOW) by KIT localization and c‐kit mutation status using an adaptive randomization scheme. Results Sixty dogs were allocated to TOC and 28 to VBL. Of the dogs receiving TOC, 20% had c‐kit mutations, compared to 30% receiving VBL (P = 0.74). Overall response rates were 46% (TOC) and 30% (VBL) (odds ratio = 1.56 [0.62–3.92]; P = 0.28). Median progression‐free survival (PFS) for dogs receiving VBL was 78 days (7–1,521) and for TOC 95.5 (14–990); hazard ratio (HR) = 1.34 [0.72–2.50]; P = 0.36. Median overall survival (OS) was 241.5 days (10–1,521) for the VBL group and 159 (20–990) for the TOC group; HR = 0.80 ([0.45–1.41]; P = 0.44). Conclusions and Clinical Importance Neither PFS nor OS was significantly different between treatment groups. As the proportion of dogs with c‐kit mutations was not different between treatment groups in this population of dogs, c‐kit mutation status did not predict treatment response.

Rabacfosadine (RAB), a novel double prodrug of the acyclic nucleotide phosphonate PMEG, preferentially targets neoplastic lymphocytes with reduced off target toxicity. Historical studies have suggested that every 21-day dosing is effective with acceptable toxicity. The purpose of this study was to evaluate RAB's safety and efficacy at 2 different doses every 21 days in dogs with relapsed B-cell lymphoma. Dogs that had failed 1 doxorubicin-based chemotherapy protocol were eligible for inclusion in this prospective trial. Once enrolled, dogs were randomized to receive RAB at either 0.82 mg/kg or 1.0 mg/kg as a 30-minute IV infusion every 21 days for up to 5 treatments. Response assessment and adverse event (AE) evaluation were performed every 21 days via VCOG criteria. Fifty dogs were enrolled, with 16 treated at 0.82 mg/kg and 34 treated at 1.0 mg/kg. The overall response rate was 74%, with 45% of dogs experiencing a complete response (CR). The median progression free intervals (PFIs) were 108 days, 172 days and 203 days for all dogs, all responders, and all CRs, respectively. Response rates and PFIs were similar in both treatment groups. The incidence of AEs, dose delays, dose reductions and withdrawals were not statistically different between the 2 groups. The AEs observed were similar to those previously reported and included hematologic, gastrointestinal, dermatologic and pulmonary AEs. One dog had grade 5 pulmonary fibrosis; otherwise, AEs resolved with supportive treatment. Rabacfosadine is a generally well tolerated, effective chemotherapy option for dogs with relapsed B-cell lymphoma.

Glutathione S-transferase-theta (GSTT1) is a carcinogen detoxification enzyme, and low activity variants are associated with lymphoma in humans. We recently found a variant in the 3' untranslated region (UTR) of canine GSTT1, *101_102insT, which was predicted to change miRNA binding and was found in 5 of 17 golden retriever (GR) dogs with lymphoma but none of 14 healthy GRs. The aim of this study was to determine whether this variant led to decreased GSTT1 expression and was a discernible risk factor for lymphoma within the GR breed. On resequencing, *101_102insT appeared to be in complete linkage disequilibrium with 3 additional 3'UTR variants, leading to the inferred haplotype *3T>C; *101_102insT; *190C>A; *203T>C. In canine livers that were heterozygous for this variant haplotype, GSTT1 protein expression was significantly lower compared to the reference haplotype (densitometry .40 vs .64, P = .022), and GSTT1 transcript levels by qPCR were also significantly lower (fold difference .52, P = .012), without evidence of substantial allelic expression imbalance. The variant haplotype led to >50% decrease in expression in vitro (.31 ± .07 vs .64 ± .19; P = .019). We found no significant difference in minor allele frequencies between 71 GR dogs with lymphoma (MAF .162) and 33 healthy age-matched controls (MAF .136, P = .69). Our results indicate that the variant GSTT1 3'UTR haplotype containing *101_102insT reduces gene expression, which could lead to impaired carcinogen detoxification, but was not a detectable risk factor for lymphoma in GR dogs.

Efficacious therapies for measurable metastatic canine osteosarcoma (OSA) are generally lacking. Preliminary retrospective studies suggested that approximately 50% of dogs with measurable metastatic OSA experienced clinical benefit (objective response or clinically meaningful disease stabilisation) following toceranib (TOC) treatment. The purpose of this clinical trial was to prospectively evaluate the clinical outcome following TOC treatment in dogs with measurable pulmonary metastatic OSA. A secondary goal was to identify potential biomarkers of clinical benefit by measuring changes in plasma vascular endothelial growth factor (VEGF) and circulating regulatory T-cell (Treg) percentage. Twenty-two dogs with pulmonary metastasis from appendicular OSA having undergone previous amputation were treated prospectively with TOC. Adverse events (AEs) were common but predominantly low grade. Nine patients were withdrawn from the study prior to the week 8 assessment of response either due to progressive disease (PD), decreased quality of life or owner perceived unacceptable AEs. Of the patients evaluable for disease progression at week 8 (or earlier), 3/17 (17.6 %) had stable disease with the remainder having PD. The median progression-free survival time for all patients was 57 days (range 7-176 days) with a median overall survival time of 89 days (range 7-574 days). Plasma VEGF concentrations were significantly elevated in patients after 4 weeks of TOC treatment, but no changes were observed in percentage of Treg in peripheral blood. Overall, the results of this clinical trial do not support the use of TOC as single agent therapy for canine metastatic OSA.

Background In humans geographical differences in the incidence and presentation of various cancers have been reported. However, much of this information has not been collected in veterinary oncology. Aim The purpose of this study was to determine if a geographic difference in progression free survival exists for dogs with lymphoma treated within the US. Materials and Methods Medical records of 775 cases of canine lymphoma from 3 US regions (west, south and north), treated with CHOP chemotherapy, were retrospectively evaluated. Cases were collected from referral institutions and were required to have received at least one doxorubicin treatment and have follow up information regarding time to progression. Results Significant differences in sex (p = 0.05), weight (p = 0.049), stage (p < 0.001), immunophenotype (p =

Background Standard of care treatment for multicentric lymphoma in dogs remains doxorubicin (DOX)-based combination chemotherapy, but owners may hesitate to commit the time and financial resources to complete such a protocol, typically requiring 12–16 visits. Rabacfosadine (RAB), a double prodrug of the nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine, has substantial single-agent activity in dogs with lymphoma, and a different mechanism of action than DOX. Hypothesis/Objectives Our objective was to evaluate the efficacy and adverse effect (AE) profile of alternating doses of RAB and DOX in dogs with naive multicentric lymphoma. Animals Fifty-four dogs with previously untreated lymphoma. Methods Open-label, multicenter prospective clinical trial. Dogs received alternating RAB (1.0 mg/kg IV weeks 0, 6, 12) and DOX (30 mg/m2 IV weeks 3, 9, 15). Dogs that achieved complete response (CR) were followed by monthly evaluations. Complete clinicopathological evaluation and assessment of remission and AEs were performed every 21 days. Results The overall response rate was 84% (68%; CR; 16%; partial response [PR)]. The overall median progression-free interval (PFI) was 194 days (216 for CR and 63 for PR). Most AEs were mild and self-limiting: gastrointestinal and hematologic AEs were most common. Thirteen dogs experienced dermatologic AEs, and 2 dogs developed grade 5 pulmonary fibrosis. Conclusions and Clinical Importance Alternating RAB/DOX generally was well tolerated and resulted in PFIs comparable to standard DOX-based multi-agent protocols, with fewer treatment visits. Most adverse events were mild or moderate and self-limiting. Further studies are warranted to explore long-term outcome and other RAB chemotherapy combinations.

While current lymphoma therapies induce remission in most dogs, drug‐resistant relapse is common, creating a need for novel agents. Rabacfosadine (RAB), a double prodrug of the acyclic nucleotide phosphonate 9‐(2‐phosphonylmethoxyethel) guanine (PMEG), preferentially targets lymphoma cells with reduced systemic toxicity compared with PMEG. Previous studies evaluating RAB administered every 21 days have suggested efficacy in both naïve and relapsed subjects; however, no large studies of RAB as a single agent have been reported in previously untreated dogs with intermediate to large cell lymphoma. The purpose of this study was to evaluate the safety and efficacy of RAB in dogs with previously untreated (excluding corticosteroids) lymphoma. Sixty‐three dogs received up to five RAB treatments every 21 days (16 at 0.82 mg/kg and 47 at 1.0 mg/kg) as a 30 minutes intravenous infusion, with (n = 23) or without (n = 40) concurrent corticosteroids. Response assessment and adverse event (Ae) evaluation were performed every 21 days via Veterinary Cooperative Oncology Group (VCOG) criteria. The overall response rate was 87% (52% CR, 35% PR). The overall median progression free interval was 122 days (199 for CR, 89 for PR and 153 days for all responders). T‐cell immunophenotype and corticosteroid pre‐treatment were predictive of inferior outcomes on multivariate analysis. AEs were most commonly of gastrointestinal origin (hyporexia/diarrhoea) and generally resolved with supportive treatment and/or dosage adjustment. Three dogs experienced VCOG‐CTCAE grade 5 delayed pulmonary fibrosis. In conclusion, RAB administered every 3 weeks is generally well tolerated and demonstrates substantial antitumour activity in dogs with previously untreated intermediate to large cell lymphoma.

Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) are implicated in the progression and outcome of a variety of pathological states, from cancer to infection. Our previous work has identified three antimicrobial peptides differentially expressed by PMN-MDSCs compared to conventional neutrophils isolated from dogs, mice, and human patients with cancer. We therefore hypothesized that PMN-MDSCs in dogs with cancer possess antimicrobial activity. In the current work, we observed that exposure of PMN-MDSCs to Gram-negative bacteria (Escherichia coli) increased the expression of reactive oxygen species by the PMN-MDSCs, indicating that they are capable of initiating an anti-microbial response. Electron microscopy revealed that the PMN-MDSCs phagocytosed Gram-negative and Gram-positive (Staphylococcus aureus) bacterial species. Lysis of bacteria within some of the PMN-MDSCs suggested bactericidal activity, which was confirmed by the recovery of significantly lower numbers of bacteria of both species following exposure to PMN-MDSCs isolated from tumor-bearing dogs. Our data therefore indicate that PMN-MDSCs isolated from dogs with cancer, in common with PMNs, have phagocytic and bactericidal activity. This nexus of immunosuppressive and antimicrobial activity reveals a hitherto unrecognized function of MDSCs.

Although novel agents hold great promise for the treatment of animal neoplasia, there may be room for significant improvement in the use of currently available agents. These improvements include altered dosing schemes, novel combinations, and patient-specific dosing or selection of agents. Previous studies have identified surrogates for "individualized dose intensity,", for example, patient size, development of adverse effects, and pharmacokinetic parameters, as potential indicators of treatment efficacy in canine lymphoma, and strategies for patient-specific dose escalation are discussed. Strategies for treatment selection in individual patients include conventional histopathology, protein-based target assessment (eg, flow cytometry, immunohistochemistry, and mass spectrometry), and gene-based target assessment (gene expression profiling and targeted or global sequencing strategies). Currently available data in animal cancer evaluating these strategies are reviewed, as well as ongoing studies and suggestions for future directions.

Circulating tumor cells (CTCs) have become an established biomarker for prognosis in patients with various carcinomas. However, current ex vivo CTC isolation technologies rely on small blood volumes from a single venipuncture limiting the number of captured CTCs. This produces statistical variability and inaccurate reflection of tumor cell heterogeneity. Here, we describe an in vivo indwelling intravascular aphaeretic CTC isolation system to continuously collect CTCs directly from a peripheral vein. The system returns the remaining blood products after CTC enrichment, permitting interrogation of larger blood volumes than classic phlebotomy specimens over a prolonged period of time. The system is validated in canine models showing capability to screen 1–2% of the entire blood over 2 h. Our result shows substantial increase in CTC capture, compared with serial blood draws. This technology could potentially be used to analyze large number of CTCs to facilitate translation of analytical information into future clinical decisions., Ex vivo methods of circulating tumor cell (CTC) isolation use small blood volumes, limiting sensitivity and introducing analytical inaccuracies. The authors describe a proof-of-concept study of an in vivo aphaeresis system that continuously collects CTCs from a peripheral vein over several hours.

Canine transitional cell carcinoma (TCC) of the bladder has historically been treated with a combination of chemotherapy, cyclooxygenase inhibitors and radiation therapy. While surgery has been used to treat TCC of the bladder, its efficacy has yet to be established. Thirty-seven client owned dogs that underwent partial cystectomy +/- various nonsurgical treatments for TCC were retrospectively evaluated. The overall median progression-free interval (PFI) was 235 days and the median survival time (ST) was 348 days. Prognostic factors identified on univariate analysis significant for ST were age, tumor location, full thickness excision and frequency of piroxicam administration. Prognostic factors significant for PFI were full thickness excision and frequency of piroxicam administration. The median ST with partial cystectomy and daily piroxicam therapy, with or without chemotherapy, was 772 days. Dogs with non-trigonal bladder TCC treated with full thickness partial cystectomy and daily piroxicam (+/- chemotherapy) may have improved outcome compared to dogs treated with medical therapy.

Canine cutaneous mast cell tumor (MCT) is the most common canine skin tumor and exhibits variable biologic behavior. Signaling through the KIT receptor tyrosine kinase promotes cellular proliferation and survival and has been shown to play a role in MCT progression. Despite investigations into numerous biomarkers and the proposal of several grading schemas, no single marker or grading system can accurately predict outcome in canine MCT. The first aim of this study was to develop an immunohistochemical assay to measure phosphorylated KIT (pKIT) to investigate its association with 2 commonly used grading systems and other established prognostic markers for canine MCT. Thirty-four archived MCTs were evaluated for expression of pKIT and Ki-67, KIT localization, mitotic count, mutations in exons 8 and 11 in c-kit, and grading by the Patnaik and 2-tier systems. Expression of pKIT was significantly ( P < .05) correlated with the 2-tier grading scheme and c-kit mutation. Correlation approached significance ( P = .06) with Mitotic Index (MI) and Ki-67. An additional aim was to determine whether pKIT labeling provides a pharmacodynamic marker for predicting response to the receptor tyrosine kinase inhibitor toceranib (TOC). MCTs from 4 of 7 patients demonstrated a partial response to TOC. pKIT expression was assessed by immunohistochemistry in biopsies obtained before and 6 hours after the patients were treated with TOC. Reduced pKIT expression after TOC treatment was demonstrated in 3 of the 4 patients with a partial response compared to 1 of the 3 nonresponders. Collectively, these results demonstrate that immunohistochemical detection of pKIT may be a clinically relevant assay to evaluate the activation status of the major oncogenic pathway in canine MCT.

Heavy ions, characterized by high linear energy transfer (LET) radiation, have advantages compared with low LET protons and photons in their biological effects. The application of heavy ions within veterinary clinics requires additional background information to determine heavy ion efficacy. In the present study, comparison of the cell-killing effects of photons, protons and heavy ions was investigated in canine osteosarcoma (OSA) cells in vitro. A total of four canine OSA cell lines with various radiosensitivities were irradiated with 137Cs gamma-rays, monoenergetic proton beams, 50 keV/µm carbon ion spread out Bragg peak beams and 200 keV/µm iron ion monoenergetic beams. Clonogenic survival was examined using colony-forming as says, and relative biological effectiveness (RBE) values were calculated relative to gamma-rays using the D10 value, which is determined as the dose (Gy) resulting in 10% survival. For proton irradiation, the RBE values for all four cell lines were 1.0–1.1. For all four cell lines, exposure to carbon ions yielded a decreased cell survival compared with gamma-rays, with the RBE values ranging from 1.56–2.10. Iron ions yielded the lowest cell survival among tested radiation types, with RBE values ranging from 3.51–3.69 observed in the three radioresistant cell lines. The radiosensitive cell line investigated demonstrated similar cell survival for carbon and iron ion irradiation. The results of the present study suggest that heavy ions are more effective for killing radioresistant canine OSA cells when compared with gamma-rays and protons. This markedly increased efficiency of cell killing is an attractive reason for utilizing heavy ions for radioresistant canine OSA.

Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth and response to therapies. The current use of large panels of cell lines with associated phenotypic and genotypic information now allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. Current cell line panels with large amounts of associated drug sensitivity and genomics data are comprised of human cancer cell lines (i.e. NCI60 and GDSC). There is increased recognition of the contribution of canine cancer to comparative cancer research as a spontaneous large animal model with application in basic and translational studies. We have assembled a panel of canine cancer cell lines to facilitate studies in canine cancer and report here phenotypic and genotypic data associated with these cells.

The goals of this work are biomarker and drug discovery to advance cancer diagnostic and therapeutic strategies in humans through the study of naturally-occurring canine cancers. Many factors, including shared environment, intact host immunity, and greater cancer gene family homology between dogs and humans than mice and humans, make spontaneous canine cancers valuable complementary models of human cancer. Transcriptomic profiling utilizing RNA sequencing (RNA SEQ) of 5 canine cancers, including osteosarcoma, melanoma, B cell lymphoma, T cell lymphoma, and pulmonary carcinoma, have revealed five distinct gene co-expression models. From these unique module expression profiles, cancer-specific gene panels were derived. A similar analysis performed on existing RNA-SEQ data from human tumor samples produced cancer-specific human gene panels. Comparison of the canine and human gene panels found significant correlation (Spearman correlation > 0.6) which supports the translational relevance of naturally-occurring canine cancers to their human counterparts. Further, proteomic profiles derived from matched tumor tissue and peripheral blood samples mirror those of the tumor transcriptome, demonstrating these cancer-specific gene panels and their encoded proteins may represent robust canine diagnostic biomarker and/or therapeutic target candidates. Therapeutic hypotheses associated with each cancer specific gene panel were derived through matching of drugs documented to alter expression of panel genes in opposition to that exhibited by each cancer type. We identified 60 candidate drugs and screened them against a panel of canine cancer cell lines, finding 40 drugs that inhibited cell growth by 75% or more. Three or more active compounds were found for each cell line. From these 40 active compounds we then derived 30 synergistic drug combinations with the requirement that that they alter two or more panel genes in opposition to that exhibited by each cancer type. Additional studies to document drug synergism are underway and those confirmed as such will be considered good drug combination candidates for future canine clinical trials. Biomarker and drug combination candidates that perform well in canine clinical trials will then be considered for human trials. This work exemplifies the type of approach meant to further establish the comparative relevance of canine to human cancer and provide opportunities to explore hypotheses related to detection and treatment in both species. Citation Format: Amy Kathleen LeBlanc, Christina N. Mazcko, Matthew Breen, Rachael Thomas, Douglas H. Thamm. A comparative oncology approach to biomarker and drug discovery for cancer diagnosis and treatment in dogs and humans [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6134.

Canine cutaneous mast cell tumour (MCT) is the most common malignant skin tumour in dogs and can exhibit variable biologic behaviour. Dysregulated signalling through the receptor tyrosine kinase (RTK) KIT can promote cell proliferation and survival, and assessment of its dysregulation via detection of activating c-kit gene mutations or assessment of KIT protein localization is associated with multiple features of malignancy. The aim of the current study was to use a previously validated immunohistochemical (IHC) assay to directly measure phosphorylated KIT (pKIT) in order to investigate its association with other established prognostic markers, response to therapy, progression free interval (PFI) and overall survival time (OST) in dogs treated medically for measurable MCT. Tumour tissue from 74 dogs enrolled in a prospective study comparing toceranib and vinblastine for MCT treatment were evaluated for pKIT immunoreactivity. pKIT was variably expressed, with some degree of positivity observed in 49/74 cases (66%). pKIT immunoreactivity was significantly associated with aberrant KIT localization, high mitotic index and high histologic grade. On univariate analysis, pKIT immunoreactivity predicted shorter PFI and OST in the entire patient population as well as shorter PFI in the toceranib treated group, and was the sole predictive factor for OST upon multivariate analysis, while mitotic index was the sole independent predictive factor for PFI. These results demonstrate that IHC detection of pKIT correlates with several features of aggressive behaviour, and may confer information that is complementary to other prognostic factors. However, the role of pKIT in predicting outcome needs to be studied further before recommendations can be made for its routine use.

One of the primary objectives of the Oncology-Pathology Working Group (OPWG), a joint initiative of the Veterinary Cancer Society and the American College of Veterinary Pathologists, is for oncologists and pathologists to collaboratively generate consensus documents to standardize aspects of and provide guidelines for oncologic pathology. Consensus is established through critical review of peer-reviewed literature relevant to a subgroup's particular focus. Subsequent acceptance and approval of the document by the OPWG membership at large establishes consensus. The intent of this publication is to help educate practitioners and pathologists on the value of diagnostics related to the KIT receptor tyrosine kinase for canine cutaneous mast cell tumours and to provide a guide for the use of these tests in veterinary medicine. This document represents the opinions of the OPWG and the authors and does not constitute a formal endorsement by the American College of Veterinary Pathologists or the Veterinary Cancer Society.

Osteosarcoma is the most common paediatric primary bone malignancy. The major cause of death in osteosarcoma is drug-resistant pulmonary metastasis. Previous studies have shown that thioredoxin reductase 2 is a driver of metastasis in osteosarcoma and can be inhibited by auranofin (AF). Moreover, studies have shown that AF significantly reduces pulmonary metastases in xenotransplant models. Here, we describe a phase I/II study of AF in canine osteosarcoma, a well-recognized spontaneous model of human osteosarcoma. We performed a single-arm multicentre pilot study of AF in combination with standard of care (SOC) (amputation + carboplatin). We recruited 40 dogs to the trial and used a historical SOC-only control group (n = 26). Dogs >15 kg received 9 mg AF q3d PO and dogs

Interest in the use of targeted microbubbles for ultrasound molecular imaging (USMI) has been growing in recent years as a safe and efficacious means of diagnosing tumor angiogenesis and assessing response to therapy. Of particular interest are cloaked microbubbles, which improve specificity by concealing the ligand from blood components until they reach the target vasculature, where the ligand can be transiently revealed for firm receptor-binding by ultrasound acoustic radiation force pulses. Herein, a bio-orthogonal “click” conjugation chemistry is introduced to decorate the surface of cloaked 4–5-μm-diameter microbubbles as part of a sterile and reproducible production process. Azido-functionalized antagonists for the angiogenic biomarkers αVβ3 integrin (cRGD) and VEGFR2 (A7R) proteins were conjugated to bimodal-brush microbubbles via strain-promoted [3 + 2] azide–alkyne cycloaddition (SPAAC) click chemistry. Ligand conjugation was validated by epifluorescent microscopy, flow cytometry, and Fourier-tra...

Metastatic cancer cells for many cancers are known to have altered cytoskeletal properties, in particular to be more deformable and contractile. Consequently, shape characteristics of more metastatic cancer cells may be expected to have diverged from those of their parental cells. To examine this hypothesis we study shape characteristics of paired osteosarcoma cell lines, each consisting of a less metastatic parental line and a more metastatic line, derived from the former by in vivo selection. Two-dimensional images of four pairs of lines were processed. Statistical analysis of morphometric characteristics shows that shape characteristics of the metastatic cell line are partly overlapping and partly diverged from the parental line. Significantly, the shape changes fall into two categories, with three paired cell lines displaying a more mesenchymal-like morphology, while the fourth displaying a change towards a more rounded morphology. A neural network algorithm could distinguish between samples of the less metastatic cells from the more metastatic cells with near perfect accuracy. Thus, subtle changes in shape carry information about the genetic changes that lead to invasiveness and metastasis of osteosarcoma cancer cells., Summary: Human and murine invasive osteosarcoma cancer cell lines, developed by selection in vivo from a less invasive parental line, show distinguishable differences in shape from the parental line that fall into two categories: more mesenchymal or more amoeboid.

Standard of care treatment of dogs with multicentric lymphoma includes combination chemotherapy with cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP); however, owners may be hesitant to commit the resources necessary to complete a lengthy, multi-drug protocol. One hundred thirty-four client-owned dogs with multicentric lymphoma were treated with a 15-week CHOP chemotherapy protocol. The overall response rate was 98% with 104 dogs experiencing a complete response (CR). The median progression-free survival (PFS) time for all dogs was 176 days, and the median disease-specific overall survival time was 311 days. Prognostic factors identified on multivariate analysis as significant for PFS included substage, immunophenotype, hospitalization for adverse events, need for dose reduction, presence of neutrophilia at diagnosis, presence of anemia and experiencing a CR as best response to therapy. In conclusion, this protocol may be a viable alternative to CHOP protocols using a larger number of treatments.

Author(s): Burton, JH; Venable, RO; Vail, DM; Williams, LE; Clifford, CA; Axiak-Bechtel, SM; Avery, AC; Thamm, DH | Abstract: BackgroundNonresectable mast cell tumors (MCT) in dogs remain a therapeutic challenge, and investigation of novel combination therapies is warranted. Intermittent administration of tyrosine kinase inhibitors (TKI) combined with cytotoxic chemotherapy may effectively chemosensitize canine MCT while decreasing cost and adverse effects associated with either agent administered as monotherapy.Hypothesis/objectivesThe primary study objectives were to (1) identify the maximally tolerated dose (MTD), (2) determine the objective response rate (ORR) and (3) describe the adverse event profile of pulse-administered toceranib phosphate (TOC) combined with lomustine.AnimalsForty-seven client-owned dogs with measurable MCT.MethodsToceranib phosphate was given PO on days 1, 3 and 5 of a 21-day cycle at a target dosage of 2.75 mg/kg. Lomustine was given PO on day 3 of each cycle at a starting dosage of 50 mg/m(2) . All dogs were concurrently treated with diphenhydramine, omeprazole, and prednisone.ResultsThe MTD of lomustine was established at 50 mg/m(2) when combined with pulse-administered TOC; the dose-limiting toxicity was neutropenia. Forty-one dogs treated at the MTD were evaluable for outcome assessment. The ORR was 46% (4 complete response, 15 partial response) and the overall median progression-free survival (PFS) was 53 days (1 to g752 days). On multivariate analysis, variables significantly associated with improved PFS included response to treatment, absence of metastasis, and no previous chemotherapy.Conclusions and clinical importanceCombined treatment with pulse-administered TOC and lomustine generally is well tolerated and may be a reasonable treatment option for dogs with unresectable or metastatic MCT.

Canine malignant melanoma, a significant cause of mortality in domestic dogs, is a powerful comparative model for human melanoma, but little is known about its genetic etiology. We mapped the genomic landscape of canine melanoma through multi-platform analysis of 37 tumors (31 mucosal, 3 acral, 2 cutaneous, and 1 uveal) and 17 matching constitutional samples including long- and short-insert whole genome sequencing, RNA sequencing, array comparative genomic hybridization, single nucleotide polymorphism array, and targeted Sanger sequencing analyses. We identified novel predominantly truncating mutations in the putative tumor suppressor genePTPRJin 19% of cases. NoBRAFmutations were detected, but activatingRASmutations (24% of cases) occurred in conserved hotspots in all cutaneous and acral and 13% of mucosal subtypes.MDM2amplifications (24%) andTP53mutations (19%) were mutually exclusive. Additional low-frequency recurrent alterations were observed amidst low point mutation rates, an absence of ultraviolet light mutational signatures, and an abundance of copy number and structural alterations. Mutations that modulate cell proliferation and cell cycle control were common and highlight therapeutic axes such as MEK and MDM2 inhibition. This mutational landscape resembles that seen inBRAFwild-type and sun-shielded human melanoma subtypes. Overall, these data inform biological comparisons between canine and human melanoma while suggesting actionable targets in both species.AUTHOR SUMMARYMelanoma, an aggressive cancer arising from transformed melanocytes, commonly occurs in pet dogs. Unlike human melanoma, which most often occurs in sun-exposed cutaneous skin, canine melanoma typically arises in sun-shielded oral mucosa. Clinical features of canine melanoma resemble those of human melanoma, particularly the less common sun-shielded human subtypes. However, whereas the genomic basis of diverse human melanoma subtypes is well understood, canine melanoma genomics remain poorly defined. Similarly, although diverse new treatments for human melanoma based on a biologic disease understanding have recently shown dramatic improvements in outcomes for these patients, treatments for canine melanoma are limited and outcomes remain universally poor. Detailing the genomic basis of canine melanoma thus provides untapped potential for improving the lives of pet dogs while also helping to establish canine melanoma as a comparative model system for informing human melanoma biology and treatment. In order to better define the genomic landscape of canine melanoma, we performed multi-platform characterization of 37 tumors. Our integrated analysis confirms that these tumors commonly contain mutations in canine orthologs of human cancer genes such asRAS,MDM2, andTP53as well mutational patterns that share important similarities with human melanoma subtypes. We have also found a new putative cancer gene,PTPRJ, frequently mutated in canine melanoma. These data will guide additional biologic and therapeutic studies in canine melanoma while framing the utility of comparative studies of canine and human cancers more broadly.

OBJECTIVES/SPECIFIC AIMS: Determine PTL’s mechanism(s) of action in a panel of canine hematopoietic cell lines; this will enable us to 1) verify that PTL is working as expected and 2) rationally select combination therapeutics. Characterize the in vitro sensitivity of canine hematopoietic cell lines to PTL in combination with other chemotherapeutic agents. Determine immunohistochemical NFκB expression in tissue microarrays of spontaneous canine neoplasms and correlate with outcome-linked data. Characterize the in vivo sensitivity of canine hematopoietic cell lines to PTL using a murine xenograft model. METHODS/STUDY POPULATION: Growth inhibition assays were performed using a panel of canine mast cell, histiocytic sarcoma, lymphoma, and leukemia cell lines, with PTL alone or in combination with redox-perturbing standard-of-care therapeutics. Cell death was assessed using flow cytometry. Immunofluorescence and immunoblotting were used to assess NFκB localization and phosphorylation of NFκB p65 (transcriptional activation), respectively. Intracellular glutathione with and without PTL and combination chemotherapeutics will be assessed spectrophotometrically. Archived spontaneous canine tumors will be evaluated immunohistochemically (IHC) for increased NFκB pathway activation relative to normal control tissues. Nude mice will receive intravenous, intraperitoneal, or subcutaneous injections of canine HS cells and will be treated with PTL or with PTL in combination with standard-of-care chemotherapeutics. RESULTS/ANTICIPATED RESULTS: Results: All immortalized canine cell lines evaluated are sensitive to PTL therapy and undergo dose-dependent apoptosis following exposure to drug. PTL exposure leads to inhibition of NFκB, as evidenced by immunofluorescent nuclear exclusion and decreased p65 phosphorylation. Some chemotherapeutics appear to synergize with PTL in vitro. Anticipated results: We expect to find increased IHC NFκB pathway activation in malignantly transformed tissues relative to controls. We expect standard-of-care therapeutics to synergize with PTL in vivo based on preliminary in vitro data. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will determine whether PTL therapy may be beneficial in dogs with a variety of hematopoietic neoplasms, either alone or in combination with other therapeutics that are currently in clinical use. Dogs with mast cell or histiocytic neoplasia are an excellent model for rare and deadly human diseases, which may also benefit from PTL therapy.

Lymph node metastasis in dogs with mast cell tumour has been reported as a negative prognostic indicator; however, no standardized histological criteria exist to define metastatic disease. The primary aim of this study was to determine whether different histological patterns of node-associated mast cells correlate with clinical outcome in dogs with mast cell tumour. A secondary goal was to propose a criteria-defined classification system for histological evaluation of lymph node metastasis. The Colorado State University Diagnostic Medicine Center database was searched for cases of canine mast cell tumours with reported lymph node metastasis or evidence of node-associated mast cells. Additional cases were obtained from a clinical trial involving sentinel lymph node mapping and node extirpation in dogs with mast cell neoplasia. Forty-one cases were identified for inclusion in the study. Demographic data, treatment and clinical outcome were collected for each case. Lymph nodes were classified according to a novel classification system (HN0-HN3) based on the number of, distribution of, and architectural disruption by, nodal mast cells. The findings of this study indicate that characterization of nodal mast cells as proposed by this novel classification system correlates with, and is prognostic for, clinical outcome in dogs with mast cell tumours.

A male castrated Golden Retriever was presented for evaluation of a large mass over the left shoulder extending to the lower part of the neck that had been present for an extended period of time, but had a recent history of rapid growth. Previous aspirates of the mass were consistent with a lipoma. The mass was surgically excised and was diagnosed as an extraskeletal osteosarcoma based on histopathology. After surgery, the dog was initiated on a chemotherapy protocol with carboplatin and metronomic cyclophosphamide. He became neutropenic, anemic, and thrombocytopenic 14 days after the carboplatin treatment was administered. The neutropenia resolved, but the anemia and thrombocytopenia progressed. A bone marrow aspirate revealed erythroid hypoplasia, myeloid hyperplasia with a predominance of early precursors, and a subset of cells that made up 20% of the total population that were reported as bizarre and unclassifiable. These cells were discrete in nature and were thought to be hematopoietic in origin. The dog was euthanized due to deterioration of the clinical condition. On postmortem examination, widespread metastasis involving the lungs, liver, kidney, heart, and bone marrow was found. Histopathology of the tumor lesions determined 2 distinct malignant populations of liposarcoma and osteosarcoma, consistent with malignant mesenchymoma. However, the possibility of 2 separate neoplastic processes cannot be definitively excluded. This is the first report of bone marrow metastasis of a malignant mesenchymoma in a dog.

Canine lymphoma (LSA) and osteosarcoma (OS) have high mortality rates and remain in need of more effective therapeutic approaches. Survivin, an inhibitor of apoptosis (IAP) family member protein that inhibits apoptosis and drives cell proliferation, is commonly elevated in human and canine cancer. Survivin expression is a negative prognostic factor in dogs with LSA and OS, and canine LSA and OS cell lines express high levels of survivin. In this study, we demonstrate that survivin downregulation in canine LSA and OS cells using a clinically applicable locked nucleic acid antisense oligonucleotide (EZN-3042, Enzon Pharmaceuticals, Piscataway Township, NJ, USA) inhibits growth, induces apoptosis and enhances chemosensitivity in vitro, and inhibits survivin transcription and protein production in orthotopic canine OS xenografts. Our findings strongly suggest that survivin-directed therapies might be effective in treatment of canine LSA and OS and support evaluation of EZN-3042 in dogs with cancer.

Background: ITK is a non-receptor tyrosine kinase that modifies T cell receptor (TCR) signaling. Mice deficient in ITK, but not resting lymphocyte kinase (RLK), exhibit defects in Th2 differentiation while retaining the ability to differentiate into Th1 cells and secrete IFNγ. Combined disruption of ITK and RLK in mice leads to more severe T cell functional defects compared to disrupting ITK alone, and paradoxically allows for normal Th2 responses. Thus, selective pharmacologic inhibition of ITK versus RLK is necessary to inhibit Th2 responses without affecting Th1-dependent immunity. ITK is widely expressed in T cell malignancies, and activation of ITK upregulates GATA-3, a transcription factor that drives Th2 differentiation and is associated with poor survival. Here we report the discovery and characterization of CPI-818, an irreversible inhibitor of ITK. CPI-818 is highly selective for ITK over RLK allowing for an assessment of pure ITK inhibition on normal and malignant T cells. Results: CPI-818 irreversibly inhibited ITK (IC502.3 nM) with >100-fold selectivity over RLK (430 nM) and BTK (850 nM). The mechanism of ITK inhibition involves covalent binding to CYS-442 confirmed by mass spectrometry. Irreversible inhibition of ITK in vitroand in vivowas demonstrated using an active site competitive probe. CPI-818 inhibited anti-CD3/28 induced phosphorylation of ERK (T202/Y204) and PLCγ (Y783) in PMBCs, and inhibited IL2 secretion in Jurkat T cells (IC5075 nM). CPI-818 demonstrated dose dependently inhibition of TCR-induced proliferation of malignant T cells from Sezary Syndrome patients. In mice orally treatedwith CPI-818 an increase in the ratio of IFNγ/IL-4 (p Citation Format: James W. Janc, Craig M. Hill, Patrick P. Ng, Andrew N. Hoston, Antonett Madriaga, Trang P. Dao-Pick, Kitman S. Yeung, Ryan Hudson, Anne-Marie Beausoleil, Erin Bradley, Erik Verner, Douglas H. Thamm, Richard A. Miller, Joseph J. Buggy. CPI-818: A selective inhibitor of interleukin-2-inducible T-cell kinase (ITK) that inhibits T-cell receptor signaling, promotes Th1 skewing, and achieves objective tumor responses when administered to dogs with T cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1313.

Analysis of circulating tumor biomarkers, designated “liquid biopsies” are less invasive and lower risk to the patient and might replace or complement standard tissue biopsies to determine prognosis, predict benefit from specific therapies, or monitor patients with cancer. Circulating tumor cells (CTCs) are extremely rare, with only 1-10 CTCs per mL of blood. Current technologies typically process 1-10 mL of blood ex vivo. We report an in vivo, indwelling CTC-capture device that is analogous to a Holter monitor used to interrogate cardiac rhythm abnormalities over long periods of time. The system incorporates a dual lumen intravenous catheter that directs blood from the vein into a small (~ the size of a wristwatch) capture carriage by virtue of a peristaltic pump. The carriage contains a CTC capture chip which separates CTCs from whole blood based on flow differences and solid phase anti-EpCAM capture, while the remaining whole blood is returned to the venous system via the dual lumen catheter. The chip is then removed from the carriage for further enumeration and characterization of the CTCs. In a proof of principle set of experiments, the system was placed into investigational canines into which fluorescently labeled human breast cancer cells were injected intravenously. Although the vast majority of these xeno-cancer cells were rapidly removed from circulation, the system captured “CTCs” over a 2 hour time period more successfully than was seen in simultaneous blood draws for ex vivo enumeration over the same time. No adverse effects were observed in the subjects at the time of the experiments or over the succeeding 48 hours after removal of the IV system.Our system permits interrogation of a much larger volume of blood than can be processed with standard blood draws. The larger processed volume will drastically increase the number of cells isolated which in turn increases the number of downstream assays that can be performed. This study is focused on developing a high throughput microfluidic device that can detect CTCs and implementing it in a canine model. Citation Format: Kaylee Smith, Tae Hyun Kim, Costanza Paoletti, Douglas H. Thamm, Daniel F. Hayes, Sunitha Nagrath. Continuous, high throughput microfluidic device to monitor circulating tumor cells in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 444.

Autophagy is the process by which cellular material is delivered to lysosomes for degradation and recycling. There are three different types of autophagy, but macroautophagy, which involves the formation of double membrane vesicles that engulf proteins and organelles that fuse with lysosomes, is by far the most studied and is thought to have important context-dependent roles in cancer development, progression, and treatment. The roles of autophagy in cancer treatment are complicated by two important discoveries over the past few years. First, most (perhaps all) anticancer drugs, as well as ionizing radiation, affect autophagy. In most, but not all cases, these treatments increase autophagy in tumor cells. Second, autophagy affects the ability of tumor cells to die after drug treatment, but the effect of autophagy may be to promote or inhibit cell death, depending on context. Here we discuss recent research related to autophagy and cancer therapy with a focus on how these processes may be manipulated to improve cancer therapy.

BackgroundMany chemotherapy protocols have been reported for treatment of canine appendicular osteosarcoma (OSA), but outcome comparisons in a single population are lacking.ObjectiveTo evaluate the effects of protocol and dose intensity (DI) on treatment outcomes for carboplatin and doxorubicin-based chemotherapy protocols.AnimalsFour hundred and seventy dogs with appendicular OSA.MethodsA retrospective cohort study was performed comprising consecutive dogs treated (1997-2012) with amputation followed by 1 of 5 chemotherapy protocols: carboplatin 300mg/m(2) IV q21d for 4 or 6 cycles (CARBO6), doxorubicin 30mg/m(2) IV q14d or q21d for 5 cycles, and alternating carboplatin 300mg/m(2) IV and doxorubicin 30mg/m(2) IV q21d for 3 cycles. Adverse events (AE) and DI were evaluated. Kaplan-Meier survival curves and Cox proportional hazards regression were used to compare disease-free interval (DFI) and survival time (ST) among protocols.ResultsThe overall median DFI and ST were 291days and 284days, respectively. A lower proportion of dogs prescribed CARBO6 experienced AEs compared to other protocols (48.4% versus 60.8-75.8%; P=.001). DI was not associated with development of metastases or death. After adjustment for baseline characteristics and prognostic factors, none of the protocols provided a significant reduction in risk of development of metastases or death.Conclusions and clinical importanceAlthough choice of protocol did not result in significant differences in DFI or ST, the CARBO6 protocol resulted in a lower proportion of dogs experiencing AEs, which could be advantageous in maintaining high quality of life during treatment. DI was not a prognostic indicator in this study.

Background An acanthocyte is an abnormally shaped erythrocyte. In veterinary medicine, acanthocytes have historically been associated with canine hemangiosarcoma. In human medicine, acanthocytes are rarely observed with neoplastic disease and are more commonly associated with a variety of hereditary and acquired diseases. Objectives The purpose of the study was to determine what disease processes are associated with the presence of acanthocytes in the peripheral blood of dogs. Methods Medical records for dogs presented to the Veterinary Teaching Hospital of Colorado State University during January 2004 through June 2008 with acanthocytes documented in their CBCs were retrospectively reviewed. Results A total of 123 dogs were included, 66 of which were diagnosed with neoplastic disease, most commonly hemangiosarcoma (n = 12), osteosarcoma (n = 11), and lymphoma (n = 11). The remaining 57 dogs had nonneoplastic disease, most commonly observed were gastrointestinal (n = 13), musculoskeletal (n = 8), renal (n = 8), and immune-mediated diseases (n = 7). No statistically significant difference was detected between percent acanthocytes present in dogs with neoplastic and nonneoplastic diseases. Conclusion Acanthocytosis was observed with a variety of neoplastic and nonneoplastic diseases. While clearly commonly associated, the presence of acanthocytes in a blood smear should not be considered pathognomonic for hemangiosarcoma in dogs.

GS-9219, a novel prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl) guanine (PMEG) has significant activity as monotherapy in dogs with non-Hodgkin's lymphoma. Phase I trials have been initiated in humans based on the encouraging activity observed in canine lymphoma. Two new analogues of GS-9219 (GS-343074 and GS-424044) were recently produced for evaluation as potential novel antineoplastic agents against solid tumours. As a preclinical step, effect of GS-343074 and GS-424044 were evaluated against ten canine cancer cell lines for antiproliferative effect. Both analogues displayed antiproliferative activity against multiple canine cancer cell lines, although GS-343074 was more potent and of broader spectrum compared to GS-424044. Flow cytometric analysis of cells that experienced growth inhibition support apoptotic death as a mechanism of action for both analogues. On the basis of in vitro results described here, GS-343074 and GS-424044 show promise as novel anticancer agents in canine cancer.

A variety of bacteria have been used as agents and vectors for antineoplastic therapy. A series of mechanisms, including native bacterial toxicity, sensitization of the immune system and competition for nutrients, may contribute to antitumor effects. However, the antitumor effects of Proteus species have been minimally studied, and it is not clear if bacteria can alter tumor hypoxia as a component of their antineoplastic effect. In the present study, Proteus mirabilis bacteria were evaluated for the ability to proliferate and accumulate in murine tumors after intravenous injection. To further investigate the efficacy and safety of bacterial injection, mice bearing 4T1 tumors were treated with an intravenous dose of 5×107 CFU Proteus mirabilis bacteria via the tail vein weekly for three treatments. Histopathology, immunohistochemistry (IHC) and western analysis were then performed on excised tumors. The results suggested Proteus mirabilis localized preferentially to tumor tissues and remarkably suppressed the growth of primary breast cancer and pulmonary metastasis in murine 4T1 models. Results showed that the expression of NKp46 and CD11c was significantly increased after bacteria treatment. Furthermore, tumor expression of carbonic anhydrase IX (CA IX) and hypoxia inducible factor-1a (HIF-1a), surrogates for hypoxia, was significantly lower in the treated group than the control group mice as assessed by IHC and western analysis. These findings demonstrated that Proteus mirabilis may a promising bacterial strain for used against primary tumor growth and pulmonary metastasis, and the immune system and reduction of tumor hypoxia may contribute to the antineoplastic and antimetastatic effects observed.

In veterinary medical oncology, there is currently no standardized protocol for assessing response to therapy in solid tumours. The lack of such a formalized guideline makes it challenging to critically compare outcome measures across various treatment protocols. The Veterinary Cooperative Oncology Group (VCOG) membership consensus document presented here is based on the recommendations of a subcommittee of American College of Veterinary Internal Medicine (ACVIM) board-certified veterinary oncologists. This consensus paper has used the human response evaluation criteria in solid tumours (RECIST v1.1) as a framework to establish standard procedures for response assessment in canine solid tumours that is meant to be easy to use, repeatable and applicable across a variety of clinical trial structures in veterinary oncology. It is hoped that this new canine RECIST (cRECIST v1.0) will be adopted within the veterinary oncology community and thereby facilitate the comparison of current and future treatment protocols used for companion animals with cancer.