A three year old male neutered mixed breed dog presented with a mass on the right carpus and accompanying lameness. A Jamshidi bone biopsy was performed, and histopathology results were consistent with a sarcoma. The dog received oncolytic virotherapy (OV) with vesicular stomatitis virus (VSV) as part of a clinical trial in dogs with osteosarcoma (OSA). Ten days after VSV treatment, the affected limb was amputated, and histopathology was consistent with intramedullary HSA. Considering the new diagnosis, standard doxorubicin chemotherapy was prescribed. With this combination of therapies, the dog had an extended survival of more than seven years and remains alive at the time of writing. This is the first case report documenting OV given in conjunction with the standard of care for canine appendicular HSA.

Abstract Angiosarcomas are rare (fewer than 300 cases each year), highly aggressive sarcomas of blood vessel-forming cells with an unfavorable prognosis. We and others have previously reported that propranolol, a beta adrenergic receptor antagonist commonly used to treat heart disease and anxiety, increased the overall survival of patients with advanced angiosarcoma when used alone or incorporated into chemotherapy regimens. Treatment of angiosarcoma has been hampered by the relative lack of suitable models to analyze molecular responses to treatment. To overcome this hurdle and understand the vulnerability of angiosarcoma to propranolol, we capitalized on the similar morphological and clinical characteristics between human angiosarcoma and canine hemangiosarcoma, which occurs in thousands of dogs each year. The objectives for the PRO-DOX clinical trial were to assess tolerability and clinical benefit of propranolol in dogs with hemangiosarcoma when given as an adjunct to chemotherapy. We conducted a phase I study using a continuous reassessment model with three dose cohorts (0.8, 1.0, and 1.3 mg/kg PO q 8 hrs) in dogs diagnosed with stage 1 or stage 2 splenic hemangiosarcoma. Following splenectomy, propranolol was onboarded using a dose escalation strategy over 12 days prior to the start of doxorubicin (30 mg/m2 intravenously every three weeks, five rounds). Twenty dogs were enrolled and distributed across the dose cohorts based on the study design. One dog in the highest cohort experienced acute hypotension after six months on propranolol, which resolved with dose reduction. Although a statistically significant survival benefit was not seen in this study when compared to historic controls, a subset of dogs (n=8 or 40%) exceeded the expected survival of 4-6 months. Notably, three dogs diagnosed at or below age five survived over two years. Gene expression analysis of tumors obtained from the short-, mid-, and long-term survivors revealed sets of genes associated with immune responses (e.g. lymphocyte activation, positive regulation of T cell activation), antigen processing, cell adhesion, and mitochondrial metabolism in the long-term survivor group. Our findings suggest that age (< 6 years) may predict a favorable outcome, and immune mediated responses differ between the short and long-term survivors. Ongoing experiments seek to define the specific molecular properties and immune responses contributing to these outcomes, with potential implications for human angiosarcoma. Citation Format: Erin B. Dickerson, Jeremy Chacón, Brian D. Husbands, Michael S. Henson, Jaime F. Modiano, Kathleen M. Stuebner, Amber Winter, Sara Pracht, Andrea Chehadeh, Kelly Bergsrud, Caitlin Feiock, Julia Medland, Heather Scavello, Pascale C. Salah, Jennifer Mahoney, Michael O. Childress, David R. Brown, Antonella Borgatti. Propranolol may enhance long term survivorship in a subset of dogs with hemangiosarcoma when combined with doxorubicin chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB407.

Abstract Our research is (focused) on delineating the relationship of aging and cancer with the goal to develop prevention and intervention therapies. Our success towards this end is based on the ability to employ companion dogs in clinical studies, particularly given the similarities of aging and cancer in dogs and people. Here, we report results of the development of both a companion diagnostic and a potential therapeutic intervention from the canine clinical Shine On study. The Shine On study included a training set of 97 dogs in four groups, consisting of (1) healthy 2-4 year-old dogs that were skeletally mature but had not reached the age boundary where cancer risk is apparent, and three groups where dogs had pathologically confirmed (2) hemangiosarcomas, (3) other malignant cancers, or (4) benign splenic masses. It also included a test set of 209 dogs over 6 years of age with no evidence of cancer or other chronic diseases. Using the Shine On Suspicion (SOS) test, dogs could be assigned to a low-risk category, where the probability of developing cancer over the next 400 days (approximately 1/10th of a modern domestic dog’s lifespan) was less than 4%, or to a high-risk category, where the probability of developing cancer over the same time period was almost 25%, and it increased to more than 50% by 1,450 days. Not surprisingly, the relative risk of cancer in dogs that were originally assigned to the low-risk category increased in a fashion that was comparable to that seen in dogs assigned to the high-risk category within 400-600 days after testing. We have used the bispecific ligand targeted toxin, eBAT, as a strategic preventative in seven dogs to date. The drug is well tolerated and associated with favorable outcomes (extended lifespan with no cancer deaths). Ongoing experiments seek to define the identity and functional properties of putative-niche forming cells and mechanisms through which we can modify the permissive environment to delay or prevent cancer in dogs - and eventually in humans. Citation Format: Jaime F. Modiano, Ashley J. Schulte, Rose Dicovitsky, Taylor A. DePauw, Ali Khammanivong, Mitzi Lewellen, Lauren E. Burt, Daniel A. Vallera, Gary R. Cutter, Amber L. Winter, Kathleen M. Stuebner, Andrea Chehadeh, Sara Pracht, Antonella Borgatti, Michael S. Henson, Erin B. Dickerson, Christopher Ober, Kari L. Anderson, Esther Nell, Aaron L. Sarver. Risk assessment, early detection, and strategic prevention of naturally occurring cancers in companion dogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7313.

Abstract Background: Osteosarcoma a common cancer in dogs that can serve as a translational model for certain aspects of human osteosarcoma. We previously developed a serum exosome gene signature in dogs with osteosarcoma capable of detecting minimal residual disease and predicting prognosis after therapy. Our goal is to continue expanding our understanding of exosomes for liquid biopsy applications in dogs with cancer, and to leverage this information in the design of liquid biopsy tests to address risk, early detection, and management of human cancer patients. For this study, we hypothesized that dogs with cancer would have higher plasma exosome concentrations compared with healthy control dogs. Methods: Plasma exosomes were enriched from 29 dogs with cancer, including 8 dogs with osteosarcoma, and 26 healthy control dogs. Exosome size and concentration in the plasma samples were characterized with nanoparticle tracking analysis. Wilcoxon rank sum tests were performed to assess differences between groups. Results: Dogs with cancer had a higher mean exosome concentration at 1.4 x 1012 (+/- 1.1 x 1012) particles/ml, compared with 5.0 x 1011 (+/- 2.7 x 1011) particles/ml in healthy control dogs (p = 0.0046). There was no difference in exosome size between dogs with cancer (mean diameter = 99.9 nm) and healthy control dogs (107.8 nm; p = 0.16) when all cancer types were included. However, exosomes enriched from healthy control samples were found to be significantly larger when compared to the exosomes enriched from the subset of cancer dogs that had osteosarcoma (mean exosome diameter = 83.9 nm; p = 0.0065). Similarly, when comparing exosome size between dogs with osteosarcoma and dogs with other cancers, plasma exosomes were larger in the dogs with other cancers (107.4 nm; p = 0.023). Conclusions: In this population, dogs with cancer have higher plasma exosome concentrations compared with healthy control dogs. Interestingly, we also found that dogs with osteosarcoma had significantly smaller exosome size than dogs with other cancers and healthy control dogs. Though the size of exosomes from dogs with osteosarcoma was found to be smaller, it is possible that the larger exosome concentration in dogs with osteosarcoma results in a similar total exosomal mass. Ongoing work includes analysis of exosomal cargo to develop a liquid biopsy test capable of assessing risk of osteosarcoma development in dogs, and applying similar methodology to determine exosomal genes associated with prognosis in pediatric osteosarcoma. The higher exosome concentrations in dogs with cancer suggests that this feature, combined with analysis of exosomal cargo, may have utility in development of a liquid biopsy cancer test in dogs. Citation Format: Kelly M. Makielski, Jaron M. Magstadt, Courtney H. Labe, Ali Khammanivong, Meagan Wojtysiak, Kyle Duval, Mitzi Lewellen, Amber Winter, Kelly Reid, Andrea Chehadeh, Michelle Buettner, Caitlin Feiock, Aaron K. Rendahl, Gary R. Cutter, Logan G. Spector, Brenda J. Weigel, Jaime F. Modiano. Dogs with cancer have higher plasma exosome concentrations compared with healthy dogs in the COED (canine osteosarcoma early detection) study [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B003.

Abstract Canine osteosarcoma is a common disease that shares many clinical and biological features with human osteosarcoma. We have previously demonstrated the potential of using mRNA signatures derived from exosome cargo to detect minimal residual disease in dogs undergoing treatment for osteosarcoma. Our goal is to continue exploring the potential of exosomes in cancer diagnosis and treatment by understanding how exosomes contribute to the biology of osteosarcoma. For this study, we compared exosome concentrations in the plasma of 26 healthy dogs and 29 dogs with various cancers, including 8 dogs with osteosarcoma. Dogs with cancer had higher exosome concentrations than the healthy dogs (p=0.0007), as did the subset of dogs with osteosarcoma. Specifically, the average exosome concentration in the samples from healthy dogs was 5x1011 (+/-2.72x1011) particles/mL, with a normal distribution ranging from 7.22x1010 to 1x1012, whereas the average exosome concentration in the samples from dogs with osteosarcoma was 1.14x1012 (+/-1.41x1012) particles/mL, with a non-parametric distribution ranging from 1.16x1011 to 4.4x1012. Ongoing work includes expanding the data set with archival samples, as well as evaluating potential relationships between exosome concentrations and disease-free survival in dogs with osteosarcoma. In conclusion, the difference in plasma exosome concentrations between healthy dogs and dogs with osteosarcoma, and specifically the heterogeneous distribution of exosomes in the plasma of dogs with osteosarcoma suggests that this could be one feature used in combination with analyses of exosome cargo to aid in prognosis and manage treatment of patients with osteosarcoma. Citation Format: Jaron M. Magstadt, Courtney H. Labé, Meagan Wojtysiak, Kyle DuVal, Ali Khammanivong, Lauren J. Mills, Amber Winter, Caitlin Feiock, Kelly Reid, Mitzi Lewellen, Logan G. Spector, Brenda J. Weigel, Jaime F. Modiano, Kelly M. Makielski. Plasma exosome concentrations in healthy canines and dogs with osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A038.

EGFR-targeted therapies are efficacious, but toxicity is common and can be severe. Urokinase type plasminogen activator receptor (uPAR)-targeted drugs are only emerging, so neither their efficacy nor toxicity is fully established. Recombinant eBAT was created by combining cytokines EGF and uPA on the same single-chain molecule with truncated Pseudomonas toxin. Its purpose was to simultaneously target tumors and their vasculature in the tumor microenvironment. In prior studies on mice and dogs, the drug proved efficacious. Here, we report the safety of eBAT in normal wildtype, uPAR knockout, and immunoreplete and immunodeficient tumor-bearing mice, as well as in dogs with spontaneous sarcoma that more closely mirror human cancer onset. In immunocompetent mice, tumor-bearing mice, uPAR knockout mice, and mice receiving species-optimized eBAT, toxicities were mild and self-limiting. Likewise, in dogs with life-threatening sarcoma given dosages found to be biologically active, eBAT was well tolerated. In mice receiving higher doses, eBAT was associated with dose-dependent evidence of liver injury, including portal biliary hyperplasia, oval cell proliferation, lymphoplasmacytic inflammation, periportal hepatocellular microvesicular change, hemorrhage, necrosis, and apoptosis. The results support continuing the clinical development of eBAT as a therapeutic agent for individuals with sarcoma and other cancers.

Abstract Companion dogs have a lifetime cancer risk similar to that of humans. This excess risk, as compared to what is seen in other species, seems to be due to our recent success in outliving our evolutionarily adapted lifespan. For cancers to develop, a cancer-permissive environment must be present, which may exist months or even years before a tumor becomes detectable. We have developed the Shine On Suspicion (SOS) test; a flow cytometry based test to detect these changes that lead to a cancer-permissive environment. To make this test actionable, we combined it with eBAT, a novel bispecific ligand-targeted toxin that targets emerging cancer cells and the inflammatory niche to disrupt cancer formation. The Shine On Study used machine- learning algorithms to determine cancer risk based on a training set of 97 dogs with known conditions and a test set of 209 adult dogs with no evidence of cancer or other chronic diseases. Dogs assigned to the low-risk category had a less than 4% chance of developing cancer within 400 days after testing, whereas dogs assigned to the high-risk category had a nearly 25% chance of developing cancer in that timeframe. By 1,450 days, more than 50% of dogs assigned to the high-risk category developed cancer. Surprisingly, the SOS test’s specificity varied by breed. In Boxers and Portuguese Water Dogs the test accurately predicted the risk of hemangiosarcoma and other malignancies, whereas for Golden Retrievers, the test accurately predicted the risk for hemangiosarcoma, but no significant difference in the development of other cancers was observed between high-risk and low-risk groups. This suggests that the contribution of the cancer-permissive environment detected by the SOS test interacts with heritable factors, i.e., those defined by ancestry in creating the overall cancer risk state. To date, eBAT interception has been administered to 9 dogs, with only 1 potentially developing cancer 2 years after treatment. The drug is well-tolerated and shows promising outcomes. Future research aims to refine the SOS test, understand breed-specific responses, and explore the identity and function of niche- forming cells to improve cancer prevention strategies and their applicability to humans. Citation Format: Ashley J Schulte, Taylor A DePauw, Ali Khammanivong, Mitzi Lewellen, Lauren Burt, Rose Dicovitsky, Amber L Winter, Kathleen M Stuebner, Andrea Chehadeh, Sara Pracht, Christopher Ober, Kari L Anderson, Esther Nell, Antonella Borgatti, Michael S Henson, Erin B Dickerson, Aaron L Sarver, Daniel A Vallera, Gary R Cutter, Jaime F Modiano. Could ancestry play a role in cancer development? Insights from a risk assessment and early detection test of naturally occurring cancers in companion dogs [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A066.

Detailed Methods for Immunohistochemistry Detailed Description of the Canine Clinical Study eBAT Pharmacokinetics and Neutralizing Antibody Assays Supplementary Table 1. Correlation between patient covariates and death Supplementary Table 2: Patient information (TMA) Supplementary Table 3: Cox regression analysis for time-to-progression Supplementary Figure 1. Survival probability based on EGFR Supplementary Figure 2. Quantification of EGFR and uPAR expression in normal canine tissues and in canine hemangiosarcoma Supplementary Figure 3. CONSORT diagram for SRCBST study

Osteosarcoma is a devastating bone cancer that disproportionally afflicts children, adolescents, and young adults. Standard therapy includes surgical tumor resection combined with multiagent chemotherapy, but many patients still suffer from metastatic disease progression. Neoadjuvant systemic oncolytic virus (OV) therapy has the potential to improve clinical outcomes by targeting primary and metastatic tumor sites and inducing durable antitumor immune responses. Here we described the first evaluation of neoadjuvant systemic therapy with a clinical-stage recombinant oncolytic Vesicular stomatitis virus (VSV), VSV-IFNβ-NIS, in naturally occurring cancer, specifically appendicular osteosarcoma in companion dogs. Canine osteosarcoma has a similar natural disease history as its human counterpart. VSV-IFNβ-NIS was administered prior to standard of care surgical resection, permitting microscopic and genomic analysis of tumors. Treatment was well-tolerated and a 'tail' of long-term survivors (~35%) was apparent in the VSV-treated group, a greater proportion than observed in two contemporary control cohorts. An increase in tumor inflammation was observed in VSV-treated tumors and RNAseq analysis showed that all the long-term responders had increased expression of a T-cell anchored immune gene cluster. We conclude that neoadjuvant VSV-IFNβ-NIS is safe and may increase long-term survivorship in dogs with naturally occurring osteosarcoma, particularly those that exhibit pre-existing antitumor immunity.

Abstract The goal of this study was to evaluate safety, efficacy, and immunological activity of Vesicular Stomatitis Virus (VSV) expressing a species relevant (canine) interferon-beta (IFN-β) and the sodium iodide symporter (NIS), henceforth called VSV, in companion dogs with naturally occurring appendicular osteosarcoma. The hypothesis was that VSV would track to the tumor, induce oncolysis, and lead to enhanced infiltration of immune cells, expansion of virus- and tumor specific T-cell clones, and improved survival outcomes.The first 15 dogs enrolled in the VSV Immunotherapy and Genomics of Osteosarcoma Research (VIGOR) study received a single iv dose of VSV (1x109 TCID50/kg) 10 days before amputation and adjuvant carboplatin chemotherapy. The final 13 dogs were randomized in a double-blinded fashion to receive either a single dose of VSV or placebo. Outcome data were compared to those from an historical control group of 57 dogs with appendicular osteosarcoma treated between 2006 and 2018 using the standard of care of alone. VSV treatment was associated with mild systemic effects (fever and inflammation), which were self-limiting and caused no symptomatology. Alterations in the tumor microenvironment (micronecrosis, increased fibrosis, and increased inflammation) were more common and more pronounced in VSV treated dogs than in placebo-treated dogs.Reduction in the expression of co-regulated cell cycle gene clusters in tumors after VSV-treatment was observed in dogs with longer survival. Conversely, increased expression of co-regulated immune/inflammatory gene clusters was observed in both VSV-treated dogs and in placebo-treated dogs, suggesting this might be, at least partly, due to the biopsy procedures. Assessment of changes in the clonal composition of T cells within the tumors, draining lymph nodes, and systemic circulation is in progress. No differences were evident in median event-free or overall survival times among the different groups, although aspects of the study design and sample size could obscure differences. Intriguingly, a “tail” of nine dogs (41%) that were long-term survivors (>450 days) was apparent in the VSV-treated group. All but one of these long-term survivors had evidence of inflammation in the pre-treatment tumor samples or showed increased inflammation in the post-treatment samples. We conclude that neoadjuvant VSV has an excellent safety profile and provides a survival benefit for a subset of dogs with spontaneous bone cancer. Ongoing work seeks to establish distinguishing characteristics that identify those individuals most likely to benefit from this therapy, as well as to evaluate the potential to enhance this benefit through the addition of novel immunomodulatory agents as part of our efforts to translate this approach to human cancer patients. Citation Format: Kelly M. Makielski, Aaron L. Sarver, Aishwarya Sathyanarayan, Michael S. Henson, Kathleen M. Stuebner, Alexandru-Flaviu Tabaran, Ingrid Cornax, Gerard O'Sullivan, Andrea Chehadeh, Donna Groschen, Kelly Bergsrud, Lauren J. Mills, Amber L. Winter, Sara Pracht, Milcah C. Scott, Michael A. Farrar, Gary R. Cutter, Joseph S. Koopmeiners, Stephen J. Russell, Shruthi Naik, Jaime F. Modiano. Oncolytic Vesicular Stomatitis Virus expressing a species relevant interferon-beta is safe and provides a survival benefit when added as neoadjuvant to the standard of care for dogs with naturally occurring bone cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1747.

Cancer mortality has surpassed all other causes of human death in parts of the developed world. Domestic dogs and cats are the only other species where cancer has an equivalent impact in overall mortality. Rapid gains in longevity over the past two centuries are sufficient to explain the increased incidence of cancer in all three species. Not only does longevity increase the probability of exposures to environmental mutagens, but it also allows for introduction of mutations in each somatic cell replication. In this context, an increase in the incidence of cancer becomes almost inevitable because the evolutionary, cancer protective adaptations of humans, dogs, and cats are insufficient to provide protection over the 2 to 6-fold extension in lifespan achieved by these species. We believe this problem is addressable by developing tests to detect malignancies at their earliest stages and pairing these tests with strategic interventions to eliminate tumors before they form. To test this premise, we have developed a blood test to detect the presence of hemangiosarcoma in dogs, a rapidly fatal malignancy. The shorter lifespan of dogs, compared to humans, and the similar shorter latency of disease provide an ideal opportunity to assess the feasibility of our approach. The test uses flow cytometry for detection of rare, non-leukocyte nucleated cells in blood, combining 42 parameters for analysis in a machine learning environment. Samples from 126 dogs with a confirmed diagnosis of hemangiosarcoma (n=28); other cancers (n=29); benign vascular pathology of the spleen (n=27); or apparently healthy and under 4 years of age (n=41) were used for training and 10-fold cross validation. The expected sensitivity and specificity for the diagnosis of hemangiosarcoma in this training set were approximately 90% and 95%, respectively. We then used our trained models to assign a risk category to apparently healthy dogs, 6 years of age or older, from three breeds with a predisposition for hemangiosarcoma (up to 20% lifetime risk): samples from 105 golden retrievers, 52 boxers and 52 Portuguese Water Dogs (N=209) were included in the study. Our results suggest that the probability of developing hemangiosarcoma or one of the other included pathological conditions within 6-18 months of testing was less than 2% in dogs with a negative result (low risk), but \u003e90% in dogs with a positive result (high risk). We will follow the dogs in this study for the duration of their lifetime to assess the predictive value of the blood test over time. Dogs at high risk of hemangiosarcoma are eligible to receive eBAT, a drug capable of eliminating the cells that maintain and propagate the tumor, while making the environment inhospitable for tumor growth. This will provide means to explore the safety and efficacy of eBAT in the setting of prevention. This study serves as proof of concept for a new paradigm of early detection and strategic prevention to reduce the societal impact of canine and human cancers. Citation Format: Jaime F. Modiano, Taylor A. DePauw, Ali Khammanivong, Ashley J. Schulte, Amber L. Winter, Jong Kim, Kathleen Stuebner, Andrea Fahrenkrug, Daniel A. Vallera, Antonella Borgatti, Erin B. Dickerson, Michael S. Henson. Early detection for strategic prevention of a terminal canine cancer: A model to reduce the impact of cancer in our society [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 4592.

We previously reported that eBAT, an EGF-targeted angiotoxin, was safe and it improved the overall survival for dogs with splenic haemangiosarcoma when added to the standard of care in a single cycle of three administrations in the minimal residual disease setting. Our objective for the SRCBST-2 trial was to assess whether increased dosing through multiple cycles of eBAT would be well tolerated and would further enhance the benefits of eBAT. Eligibility was expanded to dogs with stage 3 haemangiosarcoma, provided that gross lesions could be surgically excised. The interval between eBAT and the start of chemotherapy was reduced, and the experimental therapy was expanded to three cycles, each administered at the biologically active dose (50 mu g/kg) on a Monday/Wednesday/Friday schedule following splenectomy, and scheduled 1 week prior to the first, second and fifth doxorubicin chemotherapy. Twenty-five dogs were enrolled; six experienced acute hypotension with two requiring hospitalization. Self-limiting elevation of ALT was observed in one dog. A statistically significant survival benefit was not seen in this study in eBAT-treated dogs compared with a Contemporary comparison group of dogs with stages 1-3 haemangiosarcoma treated with standard of care alone. Our results indicate that repeated dosing cycles of eBAT starting 1 week prior to doxorubicin chemotherapy led to greater toxicity and reduced efficacy compared with a single cycle given between surgery and a delayed start of chemotherapy. Further work is needed to understand the precise mechanisms of action of eBAT in order to optimize its clinical benefits in the treatment of canine haemangiosarcoma and other tumours. IACUC Protocols 1110A06186 and 1507-32804A.

Abstract Sarcomas differ from carcinomas in their mesenchymal origin. Therapeutic advancements have come slowly, so alternative drugs and models are urgently needed. These studies report a new drug for sarcomas that simultaneously targets both tumor and tumor neovasculature. eBAT is a bispecific angiotoxin consisting of truncated, deimmunized Pseudomonas exotoxin fused to EGF and the amino terminal fragment of urokinase. Here, we study the drug in an in vivo “ontarget” companion dog trial as eBAT effectively kills canine hemangiosarcoma and human sarcoma cells in vitro. We reasoned the model has value due to the common occurrence of spontaneous sarcomas in dogs and a limited lifespan allowing for rapid accrual and data collection. Splenectomized dogs with minimal residual disease were given one cycle of eBAT followed by adjuvant doxorubicin in an adaptive dose-finding, phase I–II study of 23 dogs with spontaneous, stage I–II, splenic hemangiosarcoma. eBAT improved 6-month survival from <40% in a comparison population to approximately 70% in dogs treated at a biologically active dose (50 μg/kg). Six dogs were long-term survivors, living >450 days. eBAT abated expected toxicity associated with EGFR targeting, a finding supported by mouse studies. Urokinase plasminogen activator receptor and EGFR are targets for human sarcomas, so thorough evaluation is crucial for validation of the dog model. Thus, we validated these markers for human sarcoma targeting in the study of 212 human and 97 canine sarcoma samples. Our results support further translation of eBAT for human patients with sarcomas and perhaps other EGFR-expressing malignancies. Mol Cancer Ther; 16(5); 956–65. ©2017 AACR.

We previously described a population of lymphoid progenitor cells (LPCs) in canine B-cell lymphoma defined by retention of the early progenitor markers CD34 and CD117 and “slow proliferation” molecular signatures that persist in the xenotransplantation setting. We examined whether valspodar, a selective inhibitor of the ATP binding cassette B1 transporter (ABCB1, a.k.a., p-glycoprotein/multidrug resistance protein-1) used in the neoadjuvant setting would sensitize LPCs to doxorubicin and extend the length of remission in dogs with therapy naïve large B-cell lymphoma. Twenty dogs were enrolled into a double-blinded, placebo controlled study where experimental and control groups received oral valspodar (7.5 mg/kg) or placebo, respectively, twice daily for five days followed by five treatments with doxorubicin 21 days apart with a reduction in the first dose to mitigate the potential side effects of ABCB1 inhibition. Lymph node and blood LPCs were quantified at diagnosis, on the fourth day of neoadjuvant period, and 1-week after the first chemotherapy dose. Valspodar therapy was well tolerated. There were no differences between groups in total LPCs in lymph nodes or peripheral blood, nor in event-free survival or overall survival. Overall, we conclude that valspodar can be administered safely in the neoadjuvant setting for canine B-cell lymphoma; however, its use to attenuate ABCB1+ cells does not alter the composition of lymph node or blood LPCs, and it does not appear to be sufficient to prolong doxorubicin-dependent remissions in this setting.

Positron Emission Tomography-Computed Tomography (PET-CT) is routinely used for staging and monitoring of human cancer patients and is becoming increasingly available in veterinary medicine. In this study, 18-fluorodeoxyglucose (18FDG)-PET-CT was used in dogs with naturally occurring splenic hemangiosarcoma (HSA) to assess its utility as a staging and monitoring modality as compared to standard radiography and ultrasonography. Nine dogs with stage-2 HSA underwent 18FDG-PET-CT following splenectomy and prior to commencement of chemotherapy. Routine staging (thoracic radiography and abdominal ultrasonography) was performed prior to 18FDG-PET-CT in all dogs. When abnormalities not identified on routine tests were noted on 18FDG-PET-CT, owners were given the option to repeat a PET-CT following treatment with eBAT. A PET-CT scan was repeated on Day 21 in three dogs. Abnormalities not observed on conventional staging tools, and most consistent with malignant disease based on location, appearance, and outcome, were detected in two dogs and included a right atrial mass and a hepatic nodule, respectively. These lesions were larger and had higher metabolic activity on the second scans. 18FDG-PET-CT has potential to provide important prognostic information and influence treatment recommendations for dogs with stage-2 HSA. Additional studies will be needed to precisely define the value of this imaging tool for staging and therapy monitoring in dogs with this and other cancers.

Abstract We conducted a prospective phase I study to evaluate safety of an orally administered Salmonella encoding IL‐2 (SalpIL2) in combination with amputation and adjuvant doxorubicin for canine appendicular osteosarcoma. Efficacy was assessed as a secondary measure. The first dose of SalpIL2 was administered to 19 dogs on Day 0; amputation was done after 10 days with chemotherapy following 2 weeks later. SalpIL2 was administered concurrent with chemotherapy, for a total of five doses of doxorubicin and six doses of SalpIL2. There were six reportable events prior to chemotherapy, but none appeared due to SalpIL2. Dogs receiving SalpIL2 had significantly longer disease‐free interval (DFI) than a comparison group of dogs treated with doxorubicin alone. Dogs treated using lower doses of SalpIL2 also had longer DFI than dogs treated using the highest SalpIL2 dose. The data indicate that SalpIL2 is safe and well tolerated, which supports additional testing to establish the potential for SalpIL2 as a novel form of adjuvant therapy for dogs with osteosarcoma.

We previously described a population of lymphoid progenitor cells (LPCs) in canine B-cell lymphoma defined by retention of the early progenitor markers CD34 and CD117 and “slow proliferation” molecular signatures that persist in the xenotransplantation setting. We examined whether valspodar, a selective inhibitor of the ATP binding cassette B1 transporter (ABCB1, a.k.a., p-glycoprotein/multidrug resistance protein-1) used in the neoadjuvant setting would sensitize LPCs to doxorubicin and extend the length of remission in dogs with therapy naïve large B-cell lymphoma. Twenty dogs were enrolled into a double-blinded, placebo controlled study where experimental and control groups received oral valspodar (7.5 mg/kg) or placebo, respectively, twice daily for five days followed by five treatments with doxorubicin 21 days apart with a reduction in the first dose to mitigate the potential side effects of ABCB1 inhibition. Lymph node and blood LPCs were quantified at diagnosis, on the fourth day of neoadjuvant period, and 1-week after the first chemotherapy dose. Valspodar therapy was well tolerated. There were no differences between groups in total LPCs in lymph nodes or peripheral blood, nor in event-free survival or overall survival. Overall, we conclude that valspodar can be administered safely in the neoadjuvant setting for canine B-cell lymphoma; however, its use to attenuate ABCB1+ cells does not alter the composition of lymph node or blood LPCs, and it does not appear to be sufficient to prolong doxorubicin-dependent remissions in this setting.

Abstract Introduction: EGFuPA is a bispecific ligand-targeted toxin consisting of epidermal growth factor (EGF) and urokinase (uPA) on the same molecule with truncated Pseudomonas exotoxin. The toxin was mutated to remove amino acids recognized by B-cells. EGFuPA is uniquely designed to simultaneously target EGF receptors (EGFR) and/or urokinase receptors (uPAR) on tumor cells and uPAR in the microenvironment. EGFuPA is safe and effective in laboratory animals and has excellent in vitro activity against sarcomas. Because EGFuPA crosses species platforms, an adaptive phase 1/2 study was undertaken using canine hemangiosarcoma (HSA) as a model of a highly aggressive, metastatic vascular sarcoma. We hypothesized that six-month survival would exceed 50% for dogs enrolled. Canine HSA is a tumor derived from blood vessel forming cells, which can overexpress both uPAR and EGFR rendering it an excellent target for the drug. It bears similarity to human angiosarcoma. Methods: Enrollment began in November 2012. Dogs with histologically confirmed, non-metastatic stage-I or II splenic HSA were eligible after splenectomy. EGFuPA was administered intravenously for one cycle on Days 1, 3, and 5. Adjuvant doxorubicin was given according to standard of care (SOC) protocols starting seven days after the last dose of EGFuPA. Results: EGFuPA was detectable in the systemic circulation 5 minutes post-infusion with clearance by 15 minutes. The drug was well tolerated with self-limiting, reversible hypovolemia at the highest dose. Thus far, 50 μg/kg appears to be the optimal dose. When compared to a historical control group of 19 dogs treated with SOC between 2008 and 2011, median survival is significantly longer for all 16 dogs treated with EGFuPA (SRCBST group, 240 vs. 129 days, p = 0.03) and for the 10 dogs in the Optimal Dose group (MS not reached, p = 0.01). Six-month survival for the Optimal Dose group was 65%. Overall survival at 400 days was 7% for the SOC group, 32.7% for the SRCBST group, and 56% for the Optimal Dose group. Outcomes were not affected by the presence of neutralizing antibodies. Conclusions: A biologically active dose of EGFuPA was established and found to be safe in companion dogs with HSA in the minimal residual disease setting. The unique nature of its bispecific targeting and the observed significant, biologic activity warrants consideration of EGFuPA for a phase 1 study for drug-refractory sarcomas in human patients. Citation Format: Antonella Borgatti, Amber Winter, Kathleen Stuebner, Joseph Koopmeiners, Jaime F. Modiano, Daniel Vallera. Preclinical evaluation of a novel bispecific targeted toxin for the treatment of sarcomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1694. doi:10.1158/1538-7445.AM2015-1694