Brian K. Flesner

The association between lymphovascular invasion (LVI) and outcomes in dogs with thyroid carcinoma is inadequately described, and a standardized definition of LVI is lacking. This study's objectives were to histopathologically and immunohistochemically characterize LVI and to evaluate associated clinical outcomes in dogs with thyroid carcinoma. Inclusion criteria were: dogs that underwent thyroidectomy for thyroid carcinoma with available histopathology samples, and a minimum of 1‐year postoperative follow‐up data or follow‐up to death. Thirty dogs with 35 distinct tumours, including 26 follicular and nine medullary, were evaluated. LVI was characterized as intracapsular endothelialized tumour emboli (without adhered fibrin) in 26 samples, intracapsular tumour emboli with adhered fibrin in seven samples, extracapsular tumour emboli without adhered fibrin in 10 samples, and extracapsular tumour emboli with endothelial disruptions and adhered fibrin in three samples. CD31 and Prox1 positive vessels contained endothelialized tumour thrombi in 17 and one sample, respectively. Extracapsular invasion with (non‐endothelialized) and without (endothelialized) associated fibrin were associated with a 10‐fold ( p = 0.001) and 3‐fold ( p = 0.013) increase in the likelihood of death, respectively. No significant difference in the likelihood of death was noted between cases of extracapsular invasion with or without fibrin ( p = 0.31). Intracapsular tumour thrombi with or without associated fibrin and non‐endothelialized tumour thrombi were not associated with outcomes in this cohort. These results support that in dogs with thyroid carcinoma, extracapsular LVI with or without associated fibrin thrombi, but not intracapsular LVI, is associated with worse outcomes. Further study is warranted on a larger population of dogs with standardized treatment and follow‐up.

Purpose: Osteosarcoma is the most common primary malignancy of the skeleton. Despite advances in imaging modalities, neoadjuvant and adjuvant chemotherapy, and limb-sparing surgery over the past decades, there has been a general lack of improvement in survival rates. Tumor recurrence and metastases are associated with a worse prognosis, and complete tumor excision is critical. The use of real-time imaging could facilitate the acquisition of tumor-free surgical margins. Spontaneous osteosarcoma in dogs is an established translational model of human osteosarcoma. Experimental Design: In this study, 12 dogs with spontaneous appendicular osteosarcoma received a cathepsin-targeted quenched activity-based probe (VGT-309) intravenously 16 to 20 hours before amputation. The limb was imaged at four levels of dissection, and the margins of near-infrared fluorescence were marked for comparison with histopathology and preoperative MRI. Results: All appendicular tumors fluoresced on cross-section of the bone, and the extent of fluorescence coincided with MRI and histopathologic margins, with variability associated with necrosis. All tumors had visible fluorescence through cortical bone, although this did not consistently reflect intramedullary extension of the tumor. Necrotic tumor regions did not fluoresce. Conclusions: These data support the safety and feasibility of VGT-309 as a tool for evaluating the tissue extent of canine osteosarcoma and spur investigation for intraoperative detection of osteosarcoma during limb-sparing surgery in humans. Due to frequent intramedullary tumor extension and inconsistent fluorescence through unaffected cortical bone, this modality is likely most appropriate for assessing residual tumor following planned resection. Caution should be exercised when significant tumor necrosis is present.

Radiation therapy (RT) is a common treatment modality for dogs with locally advanced head and neck tumors. Most dogs experience a clinical benefit secondary to RT, however, long term remissions are rare. This study evaluates the feasibility and safety profile of intratumoral CBC101 (a proprietary hydrogel-based injectable resiquimod formulation), a toll-like receptor (TLR) 7/8 agonist with immunomodulatory properties, when used in combination with radiation therapy. Three dogs with histologically confirmed head/neck cancers were prospectively enrolled. A baseline CT scan was performed. Dogs received palliative radiation therapy (8 Gy × 4) in conjunction with intratumoral CB101. A follow-up CT scan was performed at week 12 to assess tumor response and to evaluate for metastatic disease. Intratumoral CB101 was well-tolerated, feasible, and produced minimal adverse effects. Only one grade 1 adverse event was attributable to CB101; all other adverse events were expected radiation therapy side effects. The data obtained from this preliminary study will be used for further investigation into appropriate dosing and timing of intratumoral resiquimod or other TLRs, with eventual escalation into phase II and III clinical trials.

Objective The Cancer Lifetime Assessment Screening Study in Canines (CLASSiC) is a prospective, longitudinal cancer screening study, in which enrolled dogs are screened for cancer with physical exams and next-generation sequencing-based liquid biopsy testing on a serial basis. The goals of the first interim analysis, presented here, are to assess the benefits of using the OncoK9® liquid biopsy test as a cancer screening tool in a prospective clinical setting, and to demonstrate test performance for cancer detection, including preclinical detection. Subjects 726 presumably cancer-free client-owned dogs were prospectively enrolled in the study across 24 clinical sites in the US and Canada. Most subjects were at high risk of cancer at the time of enrollment based on age and/or breed. 419 dogs that were enrolled for at least one year and had at least two cancer screening study visits, or that had received a definitive or presumptive diagnosis of cancer up to the time of the interim analysis, were included in the analysis. Methods Clinical data and a blood sample were collected at each study visit (once or twice per year and when cancer was clinically suspected). Cell-free DNA extracted from plasma was tested by OncoK9® using next-generation sequencing (NGS) technology. Results 417 dogs were eligible for inclusion in the interim analysis and had classifiable outcomes, with a mean on-study duration of 422 days. Of these, 51 dogs were newly diagnosed with cancer (37 definitive, 14 presumptive), translating to a 12% (51/417) observed incidence over the study period; the liver, skin, bone, heart, spleen, lung, and lymph node(s) were the most common anatomic locations for disease. The prospectively observed sensitivity (detection rate) of the test was 56.9% (95% CI: 42.3-70.4%) with a specificity of 98.9% (95% CI: 97.0-99.6%). The prospectively observed positive predictive value was 87.9% (95% CI: 70.9-96.0%) and the negative predictive value was 94.3% (95% CI: 91.3-96.3%). NGS-based liquid biopsy doubled the overall number of cancer cases detected in this study population (from 25 to 51); remarkably, the detection rate for preclinical cancer was increased 4.6-fold from 12% (6/51) by routine care alone to 55% (28/51) by combining routine care with OncoK9® testing. Clinical Relevance CLASSiC is the first study to prospectively document the incidence of cancer in a predominantly high-risk canine population, and to prospectively demonstrate that the addition of NGS-based cancer screening to regularly scheduled wellness visits has the potential to substantially increase preclinical cancer detection in this population.

Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.

Pet dogs with naturally occurring cancers play an important role in studies of cancer biology and drug development. We assessed tolerability, efficacy, and pharmacokinetic/pharmacodynamic relationships with a first-in-class small molecule inhibitor of valosin-containing protein (VCP/p97), CB-5339, administered to 24 tumor-bearing pet dogs. Tumor types assessed included solid malignancies, lymphomas, and multiple myeloma. Through a stepwise dose and schedule escalation schema, we determined the maximum tolerated dose to be 7.5 mg/kg when administered orally on a 4 days on, 3 days off schedule per week for 3 consecutive weeks. Adverse events were minimal and mainly related to the gastrointestinal system. Pharmacokinetic/pharmacodynamic data suggest a relationship between exposure and modulation of targets related to induction of the unfolded protein response, but not to tolerability of the agent. An efficacy signal was detected in 33% (2/6) of dogs with multiple myeloma, consistent with a mechanism of action relating to induction of proteotoxic stress in a tumor type with abundant protein production. Clinical trials of CB-5339 in humans with acute myelogenous leukemia and multiple myeloma are ongoing.

Supplementary Data from Comparative Oncology Assessment of a Novel Inhibitor of Valosin-Containing Protein in Tumor-Bearing Dogs

Supplementary Data from Comparative Oncology Assessment of a Novel Inhibitor of Valosin-Containing Protein in Tumor-Bearing Dogs

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

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.