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Supplementary Table 3 contains a summary of the outcomes, with statistical comparisons, of the dogs enrolled in the SOC clinical trial arm. This includes the DFI and outcomes of the groups of dogs stratified by tumor location and ALP status.

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

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

Purpose:The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression.Patients and Methods:A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes.Results:There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144–237] and 282 days (95% CI, 224–383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157–217) and 280 days (95% CI, 252–332), respectively.Conclusions:In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

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

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

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

Primary pulmonary histiocytic sarcoma (PHS) is a rare form of dendritic cell or macrophage neoplasia originating within the pulmonary parenchyma. There is limited literature describing prognosis in dogs with PHS receiving curative-intent treatment consisting of surgical excision and adjuvant chemotherapy. The primary objective of this study was to report outcomes in dogs with localized PHS treated with standardized local and systemic therapy. A secondary objective was to identify prognostic factors in this population. A multi-institutional retrospective study was performed and medical records including all surgical and histopathologic reports were retrospectively reviewed. For inclusion, dogs were required to have confirmed localized PHS and they must have undergone curative-intent surgery with resection of all gross primary tumour and enlarged tracheobronchial lymph nodes; additionally, they must have received curative-intent treatment with adjuvant single-agent CCNU chemotherapy. Twenty-seven dogs from six veterinary teaching hospitals and five private practices treated from 2008-2019 were included. The overall median survival time was 432 days. Higher CCNU dose was demonstrated to have a negative impact on survival on univariate, but not multivariable, analysis. Factors that were not found to be associated with survival on univariate analysis included body weight, breed, clinical signs at the time of diagnosis, hypoalbuminaemia, tumour size, lung lobe affected, lymph node metastasis, surgical margins and CCNU dose reductions. This study supports a favourable prognosis for dogs diagnosed with localized PHS treated with curative-intent surgery in addition to adjuvant CCNU chemotherapy and suggests that multimodal treatment may be advisable to attempt to prolong survival.

Local recurrence after surgical excision of canine massive hepatocellular carcinoma (HCC) has been poorly studied in veterinary medicine with scant information published regarding risk factors for and outcome following recurrence. The aim of this case-control study was to describe the time to recurrence, evaluate potential risk factors for recurrence, and report the outcome in dogs with massive HCC. Medical records for 75 dogs who developed recurrence and 113 dogs who did not develop recurrence were reviewed. Statistical analyses were performed to determine risk factors for recurrence as well as the median time to develop recurrence and overall survival time (OS). None of the risk factors evaluated were significant for the development of recurrence. The median time to develop recurrence was 367 days (range 32-2096 days). There was no significant difference in median OS for dogs who developed recurrence vs. those who did not (851 vs. 970 days). For dogs with recurrent HCC, treatment at recurrence trended toward prolonged OS but was not significantly different from dogs not undergoing treatment at recurrence. There was no significant difference in median OS for dogs with histologically complete vs. incomplete tumour excision (990 vs. 903 days). Although specific risk factors for recurrence were not identified, elevations in liver values were noted in patients with recurrent disease and could act as a noninvasive surveillance tool. Recurrence was noted earlier in dogs who had routine post-operative surveillance (228 vs. 367 days). Routine surveillance for recurrence is recommended especially in dogs where further intervention is possible and should extend beyond 1 year. Patients with massive HCC have a good long-term prognosis regardless of incomplete excision, pulmonary metastasis, or recurrent local disease. Published version

Background: Localized splenic histiocytic sarcoma (HS) in dogs is a poorly understood disease, and could have longer survival times than disseminated or hemophagocytic HS. Understanding the clinical behavior of localized splenic HS can refine treatment recommendations. Objective: To describe the clinical characteristics and outcomes of dogs with localized splenic HS. Animals: Fourteen client-owned dogs with histologically confirmed splenic HS that received splenectomy. Methods: Multi-institutional retrospective case series—medical records of dogs with splenic HS were reviewed. Dog signalment, clinicopathologic data, primary and adjuvant treatments, and outcomes were obtained. Survival data were calculated using Kaplan-Meier analysis. Dog variables such as age, weight, platelet counts were reported using descriptive statistics. The Cox proportional hazards regression method was used to determine whether potential risk factors (weight, age, albumin level, hematocrit, and platelet count) were associated with PFI. Results: Median survival time for the dogs in this study was 427 days. Twelve dogs received adjuvant lomustine-based chemotherapy. Five dogs (35.7%) were suspected or confirmed to have developed metastatic disease. Eleven dogs died of disease, 1 dog died of unrelated cause, and 2 dogs were alive at final follow-up. Conclusions and Clinical Significance: Histiocytic sarcoma in dogs can manifest as a localized form in the spleen. Dogs with localized splenic HS treated with surgery ± chemotherapy can experience survival times over a year. Published version

Fasting has been shown to decrease chemotherapy‐associated adverse events (AEs), in part through insulin‐like growth factor (IGF‐1) reduction, and may induce a protective effect on normal cells during chemotherapy treatment in mice and people. The purpose of this study was to evaluate the effect of fasting on constitutional, bone marrow and gastrointestinal (GI) AEs, and serum glucose, IGF‐1 and insulin levels in dogs receiving vincristine. The study was a prospective, crossover clinical trial in tumour‐bearing dogs. Dogs were randomized to be fasted for 24 to 28 hours prior to and 6 hours following their first or second vincristine treatment, and fed normally for the alternate dose. A significant reduction in nausea, anorexia, lethargy and serum insulin was observed when dogs were fasted; however, no significant differences were found in other GI symptoms, neutrophil count, serum glucose or IGF‐1. Fasting prior to vincristine therapy is a safe and effective treatment modality that helped mitigate constitutional and GI AEs in tumour‐bearing dogs.

Low-grade canine cutaneous mast cell tumour (cMCT) with metastasis at the time of treatment is uncommonly reported, with few studies focusing on this specific clinical entity. The specific objective of this study was to systematically review the veterinary literature and perform a meta-analysis summarizing the clinical presentation, treatments reported and clinical outcomes from dogs with histologically low-grade cMCT and metastasis present at initial treatment. A total of 980 studies were screened with eight publications providing data on 121 dogs ultimately included. The most common treatments were surgery with adjuvant chemotherapy in 83/121 (69%) dogs; combined surgery, radiation and chemotherapy in 17/121 (14%) dogs; chemotherapy alone in 12/121 (10%) dogs and surgery alone in 7/121 (6%) dogs. Dogs with distant metastasis (n = 22) experienced significantly shorter survival compared with those with regional lymph node (RLN) metastasis (n = 99; median 194 vs 637 days; P < .01). Two variables were significantly associated with increased risk of death: presence of distant (vs RLN) metastasis (hazard ratio = 2.60; P < .01) and not receiving surgery as a component of treatment (hazard ratio = 3.79; P < .01). Risk of bias was judged to be low in terms of selection and performance bias but high in terms of detection and exclusion bias. In conclusion, dogs with cMCT and RLN metastasis can be expected to live significantly longer than those with distant metastasis, and surgery appears to have a role in extending survival of metastatic low-grade cMCT.

Cancer patients commonly develop infectious complications over the course of the disease. One thousand patients receiving treatment for an oncologic disease at a single veterinary teaching hospital were retrospectively reviewed for concurrent infections. A total of 153 confirmed bacterial infections were identified, 82 of which were abscesses or wounds, 13 of which were respiratory infections, 3 of which were ear infections, and 55 of which were urinary tract infections. It was observed that the majority of the infections were caused by bacteria that are normally associated with that specific site location. Escherichia coli was the most common pathogen linked to infections in general, but Staphylococcus pseudintermedius was a frequently identified pathogen associated with wound infections. The susceptibility to diverse antimicrobials varied with the site of infection. Eleven cases (7.1%) were caused by opportunistic infections of the site, and E. coli and Pseudomonas aeruginosa were the pathogens isolated. Those bacteria were resistant to many antibiotics but showed susceptibility to aminoglycosides, imipenem, quinolones, and polymyxin B. In conclusion, veterinary patients with cancer or those under treatment for tumors develop infections by commonly encountered bacteria in the different sites of the body, with a susceptibility to antibiotics that is not out of line from what is expected. A small subset of cases developed opportunistic infections, with microbes that were more resistant to many classes of antibiotics.

Purpose: The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression. Patients and Methods: A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes. Results: There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144–237] and 282 days (95% CI, 224–383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157–217) and 280 days (95% CI, 252–332), respectively. Conclusions: In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

Background Osteosarcoma patients often experience poor outcomes despite chemotherapy treatment, likely due in part to various mechanisms of tumor cell innate and/or acquired drug resistance. Exosomes, microvesicles secreted by cells, have been shown to play a role in drug resistance, but a comprehensive protein signature relating to osteosarcoma carboplatin resistance has not been fully characterized. Methods In this study, cell lysates and exosomes from two derivatives (HMPOS-2.5R and HMPOS-10R) of the HMPOS osteosarcoma cell line generated by repeated carboplatin treatment and recovery, were characterized proteomically by mass spectrometry. Protein cargos of circulating serum exosomes from dogs with naturally occurring osteosarcoma, were also assessed by mass spectrometry, to identify biomarkers that discriminate between good and poor responders to carboplatin therapy. Results Both cell lysates and exosomes exhibited distinct protein signatures related to drug resistance. Furthermore, exosomes from the resistant HMPOS-2.5R cell line were found to transfer drug resistance to drug-sensitive HMPOS cells. The comparison of serum exosomes from dogs with a favorable disease-free interval [DFI] of > 300 days, and dogs with Conclusions The findings of this study highlight the significance of exosomes in the potential transfer of drug resistance, and the discovery of novel biomarkers for the development of liquid biopsies to better guide personalized chemotherapy treatment.

Case summary A case of nasal adenocarcinoma as a suspected secondary malignant neoplasm following definitive radiation therapy and multiagent chemotherapy for nasal lymphoma is described. An 11-year-old spayed female domestic shorthair cat was presented for a 3-week history of progressive facial swelling located over the nasal planum and extending to the medial canthus of the right eye. The cat was previously diagnosed with nasal lymphoma and treated with chemotherapy and definitive radiation 2.5 years prior. Although a definitive diagnosis could not be obtained via cytology, recurrent lymphoma was suspected based on the cat’s history and recurrent clinical signs. A lymphoma-directed chemotherapy protocol was attempted, but no clinical response was achieved. The cat was euthanased owing to progressive clinical signs and a diagnosis of nasal adenocarcinoma was made on necropsy examination. Both the original diagnosis of nasal lymphoma and the secondary diagnosis of nasal adenocarcinoma were confirmed with immunohistochemistry. Relevance and novel information Secondary malignant neoplasm following radiation therapy is infrequently reported in the veterinary literature. In the few reports that exist, most have described sarcoma development in the dog following radiation therapy. In the present report, we describe a cat with a suspected radiation-induced nasal adenocarcinoma that developed 2.5 years after definitive radiation treatment for nasal lymphoma.

Circulating cancer exosomes are microvesicles which originate from malignant cells and other organs influenced by the disease and can be found in blood. The exosomal proteomic cargo can often be traced to the cells from which they originated, reflecting the physiological status of these cells. The similarities between cancer exosomes and the tumor cells they originate from exhibit the potential of these vesicles as an invaluable target for liquid biopsies. Exosomes were isolated from the serum of eight osteosarcoma-bearing dogs, five healthy dogs, and five dogs with traumatic fractures. We also characterized exosomes which were collected longitudinally from patients with osteosarcoma prior and 2 weeks after amputation, and eventually upon detection of lung metastasis. Exosomal proteins fraction were analyzed by label-free mass spectrometry proteomics and were validated with immunoblots of selected proteins. Ten exosomal proteins were found that collectively discriminate serum of osteosarcoma patients from serum healthy or fractured dogs with an accuracy of 85%. Additionally, serum from different disease stages could be distinguished with an accuracy of 77% based on exosomal proteomic composition. The most discriminating protein changes for both sample group comparisons were related to complement regulation, suggesting an immune evasion mechanism in early stages of osteosarcoma as well as in advanced disease.

The objective of this prospective study was to evaluate agreement and reliability of calliper-based measurements of locally invasive subcutaneous malignant tumours in dogs. Four raters measured the longest diameter of 12 subcutaneous tumours (7 soft tissue sarcomas and 5 mast cell tumours) from 11 client-owned dogs during 3 randomized, blinded measurement trials, both pre- and post-sedation. Inter- and intra-rater reliability was evaluated using intra-class correlation coefficient (ICC) and agreement was evaluated using Bland-Altman plots. Inter- and intra-rater reliability was good (ICC range of 0.8694-0.89520) and excellent (ICC range of 0.9720-0.9966), respectively. For agreement calculations, an a priori clinically relevant limit of agreement of 10 mm was set. Inter- and intra-rater agreement was unacceptable with inter-rater limits of agreement ranging from 15.9 to 55.6 mm and intra-rater limit of agreement ranging from 11.9 to 28.1 mm. Review of the measurement trial photographs revealed that calliper orientation changes were frequent, occurring in 9/12 (75%) and 8/12 (67%) pre- and post-sedation cases. No significant correlation was found between inter-rater measurement standard deviations and calliper orientation changes or dog body condition score. These findings suggest veterinarians may have poor agreement in determining the gross edge of tumours, which is expected to introduce bias and inconsistency in tumour staging, assessing response to therapy, and surgical margin planning. Due to the potential consequences for veterinary cancer patients, future studies are needed to validate the present findings.

Immune checkpoint therapy (ICT) is a front-line treatment for lung cancer; however, low mutational burden and ‘non-T cell inflamed' signatures predict poor responses to ICT in ~50% of patients. Adoptive cellular therapy (ACT) with T cells engineered to express T cell receptors (TCRs) specific for tumor-associated antigens (TAAs; proteins overexpressed by cancers) is an approach that circumvents the need for endogenous T cell responses. TCR-ACT is effective against hematologic cancers, but ACT against solid tumors is in the early stages of exploration. A deeper understanding of complex interactions between therapeutic cells and the tumor microenvironment (TME) is crucial for identifying successful strategies to enhance function and mitigate toxicity. Genetically engineered mouse models (GEMMs) achieve in situ tumor development alongside competent immune systems, recapitulating the native TME over the full spectrum of disease progression in a preclinical setting. KP GEMM lung tumors harbor few mutations, are poorly infiltrated by T cells, and are refractory to ICT, modeling intractable patient disease. We show that TAA-specific T cells recognize KP lung cancer cells and efficiently home to tumors, but rapidly lose function in the lung TME (compared to KP pancreatic tumors). Repetitive TCR-ACT can extend survival of animals with lung tumors, but animals eventually succumb to disease. These results show promise for TCR-ACT against lung cancer but highlight tissue-specific obstacles that must be overcome to enhance efficacy. Preliminary studies indicate that therapeutic T cells expressing immunomodulatory fusion proteins (IFPs) or accompanied by engineered helper T cells exhibit increased function in lung tumors, indicating that increased costimulatory signaling and T cell help can partially overcome T cell suppression in the lung TME. As we continue to leverage these models to advance our understanding of tissue-specific effects on therapeutic outcomes, these findings offer inroads to uncovering more effective therapies, with the ultimate goal of benefitting cancer patients. Citation Format: Leah Marie Schmidt, Oanh Tran, Cody Jenkins, Shannon Oda, Quintin Inman, Haley Leeper, Sasha Tan, Philip Greenberg. Effects of the lung tumor microenvironment on T cell therapy [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 1510.

Objectives To determine if total serum cholesterol concentrations were altered in dogs with osteosarcoma. To evaluate association of total serum cholesterol concentration with clinical outcomes in dogs with appendicular osteosarcoma. Materials and Methods Retrospective, multi-institutional study on 64 dogs with osteosarcoma. Control population consisted of dogs with traumatic bone fractures (n=30) and healthy patients of similar age and weight as those of the osteosarcoma cases (n=31). Survival analysis was done on 35 appendicular osteosarcoma patients that received the current standard of care. Statistical associations were assessed by univariable and multi-variable analysis. Information about age, sex, primary tumour location, total cholesterol concentration, monocytes and lymphocyte counts and alkaline phosphatase were also included. Results Total cholesterol was elevated above the reference interval (3·89 to 7·12 mmol/L) (150 to 275 mg/dL) in 29 of 64 (45·3%) osteosarcoma-bearing dogs, whereas similar elevations were found in only 3 of 30 (10%) fracture controls (P