Oral tumors are relatively common in dogs, and canine oral squamous cell carcinoma (COSCC) is the most prevalent oral malignancy of epithelial origin. COSCC is locally aggressive with up to 20% of patients showing regional or distant metastasis at the time of diagnosis. The treatment of choice most typically involves wide surgical excision. Although long-term remission is possible, treatments are associated with considerable morbidity and can negatively impact functionality and quality of life. OSCCs have substantial upregulation of the RAS-RAF-MEK-MAPK signaling axis, and we had previously hypothesized that small-molecule inhibitors that target RAS signaling might effectively inhibit tumor growth and progression. Here, we demonstrate that the MEK inhibitor trametinib, an FDA-approved drug for human cancers, substantially inhibits the growth of six COSCC cell lines established from current patient tumor samples. We further show preliminary clinical evidence that the drug is able to cause ~ 40% and ~ 80% tumor regression in two out of four patients with spontaneously occurring COSCC, a partial response according to commonly used RECIST criteria. Given the limited treatment options available and the number of dogs for which standard of care is not acceptable, these preliminary findings provide new hope that more suitable treatment options may soon enter the veterinary clinic.

A clinical trial in dogs with spontaneous osteosarcoma was performed to assess a recombinant Listeria expressing a chimeric human HER2 (ADXS31-164c) as an adjunctive vaccine strategy to prevent metastatic disease and determine immunological correlates of clinical outcome. A total of 118 dogs with appendicular osteosarcoma were recruited into a 1-arm, multicenter, prospective trial of standard of care (SOC) therapy followed by ADXS31-164c. ADXS31-164c was well tolerated, with mostly transient, low-grade side effects. Significant differences in median disease-free interval (DFI) or median overall survival (OS) of immunized dogs compared to a historical cohort of dogs receiving SOC only were not observed. Elite survivors (DFI >490 days) showed transient increases in temperature and serum cytokines, including IL-6 and TNF-α, after the first immunization compared to short-term survivors (DFI 150-235 days). However, repeat immunizations in short-term survivors led to improved and comparable pyrexic and cytokine responses to elite survivors. PBMC transcriptomic analysis following vaccinations revealed robust cytotoxic activity in elite but not short-term survivors. Although ADXS31-164c did not significantly extend DFI or OS, immune responses to ADXS31-164c distinguished elite from short-term survivors. Improvement of immune responses over sequential ADXS31-164c administrations supports a future trial design of recurrent immunizations to improve outcomes of otherwise short-term survivors.

Oral squamous cell carcinoma (OSCC) is a common and, if left untreated, deadly disease in dogs. The current standard of care involves wide-margin surgical excision of tumor tissue, which is frequently disfiguring. Surgical treatment can also be debilitating and lead to a reduced quality of life. Recent studies have demonstrated that OSCC in dogs typically shows highly elevated RAS signaling compared to healthy gingival tissue. Here, we examine the drug trametinib, which is FDA-approved for use in humans for BRAF-mutant melanomas, as an approach to treating OSCC in dogs. Domestic companion dogs (N = 20) with spontaneously occurring OSCC were recruited over a two-year period for an interventional study without concurrent controls. Dogs were prescribed 0.015 to 0.035 mg/kg trametinib daily to be given orally. Treatment was continued for 8 weeks, with examinations every 2 weeks. Health monitoring was conducted via routine bloodwork combined with at-home questionnaires. Tumor volume was assessed by caliper measurement and by computed tomography (CT) imaging. Tumor response categorization was based on R.E.C.I.S.T. criteria. Final R.E.C.I.S.T. categorization was based on CT measurement, while mid-experiment detection of progressive disease (PD) was based on caliper measurement. Dogs exited the study immediately upon determination of PD. Five dogs (25

OBJECTIVE:To evaluate the clinical benefit and adverse events following outpatient intravenous (IV) ketamine-lidocaine (KL) infusions for palliation of cancer pain in dogs and cats. METHODS:Medical records from 15 years (2008 to 2023) were searched and data reviewed retrospectively. Animals were prescribed ketamine (0.15 mg/kg/h) and lidocaine (3 mg/kg/h in dogs and 1.5 mg/kg/h in cats) diluted in 250 mL (cats or dogs < 15 kg) or 500 mL (dogs ≥ 15 kg) of 0.9% saline administered IV at 2.5 mL/kg/h for 4 to 6 hours. RESULTS:A total of 105 dogs and 9 cats (114 animals) were included. The most common tumor types treated were appendicular osteosarcoma in dogs (n = 45 [43%]) and oral squamous cell carcinoma in cats (4 [44%]). All animals received concomitant oral analgesic medications. Animals received a median of 2 KL infusions (range, 2 to 49 KL infusions). The overall clinical benefit rate was 76% following the first KL infusion, as defined by an improvement in at least 1 evaluated clinical sign. The likelihood of clinical response was improved with a ketamine infusion rate ≥ 2 µg/kg/min, total ketamine dose of ≥ 0.5 mg/kg, and lidocaine infusion rate ≥ 25 µg/kg/min. CONCLUSIONS:Outpatient IV KL infusions were well-tolerated in dogs and cats with cancer and provided a high rate of clinical benefit when combined with standard anti-cancer and analgesic therapies. CLINICAL RELEVANCE:Management of cancer pain often requires multimodal analgesia. Given the clinical benefit and minimal toxicity observed with outpatient KL infusions, KL infusions should be considered as an adjunctive treatment to use in combination with anticancer therapy, oral and topical analgesics, and interventional therapies in dogs and cats for palliation of refractory cancer pain from advanced disease.

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 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 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

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

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.

Background: Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer in dogs. Despite this, relatively few reports of this disease exist pertaining to prognostic factors and outcome. Aim: To evaluate factors associated with survival in dogs with all subtypes of HCC diagnosed on histopathology. Methods: A retrospective single institutional study was carried out on 94 client-owned dogs with a histopathologic diagnosis of HCC between 2007 and 2018 obtained by biopsy (21/94) or attempted definitive resection (73/94). Signalment, preoperative features, surgical findings, and postoperative outcomes were recorded. Associations between survival to discharge data were collected and univariable logistical regression was carried out. Kaplan–Meier survival analysis was carried out to identify negative risk factors for long-term prognosis. Results: The median survival time (MST) for all patients was 707 days (95% CI = 551–842). MST was not significantly different (p > 0.05) between patients who had suspected versus incidentally diagnosed HCC (695 vs. 775 days), between complete versus incomplete surgical margins (668 vs. 834 days), or between patients with massive subtype versus nodular/diffuse subtype (707 vs. 747 days). Logistical regression identified an association with the excision of the right medial lobe and risk of perioperative death (OR = 9.2, CI 1.5–55.9, p = 0.016). An American Society of Anesthesiologists score ≥4, disease present within the quadrate lobe, and elevated blood urea nitrogen, potassium or gamma-glutamyltransferase were identified as negative prognosticators during multivariable Cox regression. Preoperative imaging (ultrasound or CT) agreed with the surgical location in 91% of the cases. Preoperative cytology was consistent with a diagnosis of HCC in 15/32 (46.9%) cases. Conclusion: Type of diagnosis (incidental vs presumed), completeness of excision, and subtype were not associated with MST in this study. Preoperative identification of tumors within the central division may be related to a less favorable outcome. Results of preoperative cytology were not highly sensitive for identifying a malignancy.

Abstract 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.

Doxycycline has antiproliferative effects in human lymphoma cells and in murine xenografts. We hypothesized that doxycycline would decrease canine lymphoma cell viability and prospectively evaluated its clinical tolerability in client-owned dogs with spontaneous, nodal, multicentric, substage a, B-cell lymphoma, not previously treated with chemotherapy. Treatment duration ranged from 1 to 8 weeks (median and mean, 3 weeks). Dogs were treated with either 10 (n = 6) or 7.5 (n = 7) mg/kg by mouth twice daily. One dog had a stable disease for 6 weeks. No complete or partial tumor responses were observed. Five dogs developed grade 3 and/or 4 metabolic abnormalities suggestive of hepatopathy with elevations in bilirubin, ALT, ALP, and/or AST. To evaluate the absorption of oral doxycycline in our study population, serum concentrations in 10 treated dogs were determined using liquid chromatography tandem mass spectrometry. Serum levels were variable and ranged from 3.6 to 16.6 µg/ml (median, 7.6 µg/ml; mean, 8.8 µg/ml). To evaluate the effect of doxycycline on canine lymphoma cell viability in vitro, trypan blue exclusion assay was performed on canine B-cell lymphoma cell lines (17-71 and CLBL) and primary B-cell lymphoma cells from the nodal tissue of four dogs. A doxycycline concentration of 6 µg/ml decreased canine lymphoma cell viability by 80%, compared to matched, untreated, control cells (mixed model analysis, p < 0.0001; Wilcoxon signed rank test, p = 0.0313). Although the short-term administration of oral doxycycline is not associated with the remission of canine lymphoma, combination therapy may be worthwhile if future research determines that doxycycline can alter cell survival pathways in canine lymphoma cells. Due to the potential for metabolic abnormalities, close monitoring is recommended with the use of this drug in tumor-bearing dogs. Additional research is needed to assess the tolerability of chronic doxycycline therapy.

The incidence of proteinuria in humans receiving tyrosine kinase inhibitors has been well-documented. Reports of proteinuria with this class of drugs are limited in veterinary medicine. This retrospective study describes the incidence, severity, and progression of proteinuria in 55 dogs treated with toceranib phosphate, with or without concurrent glucocorticoid or NSAID (non-steroidal anti-inflammatory drug). Six dogs were proteinuric at baseline. Twelve of the 49 dogs that were not proteinuric at baseline developed proteinuria while receiving toceranib phosphate. Median urine protein: creatinine (UPC) ratio when proteinuria developed was 0.75 (range: 0.6 to 4.9). There was no association with intermittent glucocorticoid or NSAID use and development of proteinuria (P = 0.5 and P = 0.7, respectively). Overall duration of toceranib phosphate treatment ranged from 70 to 802 days in proteinuric dogs and 28 to 1285 days in non-proteinuric dogs. Our results indicate a subset of dogs receiving toceranib phosphate may develop proteinuria; careful monitoring with serial UPCs is recommended.

An In Vitro study was conducted to investigate docetaxel as a radiation sensitizer in four canine (mammary carcinoma—CMT12 and CMT25, osteosarcoma—OS2.4, and transitional cell carcinoma—PTCC), and one feline cancer cell line (oral squamous cell carcinoma—SCCF1) to provide a basis for combination therapy in clinical patients. Cells were exposed to docetaxel followed by a single dose of radiation. The percent surviving fraction was determined by MTT assay. The combination index (CI) method determined synergistic cytotoxicity for the CMT12, CMT25 and OS2.4 cell lines with median CI values of 0.35, 0.47, 0.63 respectively. The SCCF1 cell line had moderate synergistic cytotoxicity with a median CI of 0.76, while the PTCC cell line resulted in antagonistic cytoxicity with a median CI of 2.75. The results indicated that docetaxel was a radiation sensitizer in 4 out of the 5 cancer cell lines tested.

Objective-To determine the effects of lycopene with and without concurrent chemotherapeutic treatment on growth and apoptosis of canine osteosarcoma cells.Sample Population-Cell cultures of 3 established canine osteosarcoma cell lines (D17, OS 2.4, and HMPOS).Procedures-Growth curve kinetics and cell cytotoxicosis for various treatment combinations were assessed by use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Additionally, cell cycle kinetics and colony-forming soft agar assays were performed to determine the influences of lycopene on the cell cycle and anchorage-independent growth. Western immunoblotting of HMPOS cells was performed to examine signaling and apoptotic pathways implicated in lycopene-induced apoptosis.Results-Lycopene alone caused mild to pronounced attenuation of cell proliferation of all 3 cell lines as well as apoptosis in HMPOS cells but did not interfere with cell death in response to doxorubicin. Soft agar anchorage-independent growth assays revealed complete inhibition of cell proliferation in 2 of 3 osteosarcoma cell lines. Further investigation into the apoptotic response revealed activation of mitochondrial-induced apoptosis primarily through expression of truncated Bid and a decrease in protein kinase B (ie, AKT) phosphorylation.Conclusions and Clinical Relevance-Results suggested that lycopene may be beneficial during treatment of osteosarcomas. Lycopene did not negatively or positively affect survival of osteosarcoma cells during doxorubicin treatment and independently induced apoptosis in the HMPOS cell line. These findings warrant further in vitro and in vivo studies into the use of this natural compound as an adjuvant antiproliferative, proapoptotic treatment in dogs with osteosarcoma. (Am J Vet Res 2010;71:1362-1370)

Calcitriol potentiates cisplatin-mediated activity in a variety of tumor models. We examine here, the effect of calcitriol and cisplatin pre-clinically and clinically in canine spontaneous tumors through in vitro studies on tumor cells and through a phase I study of calcitriol and cisplatin to identify the maximum-tolerated dosage (MTD) of this combination in dogs with cancer and to characterize the pharmacokinetic disposition of calcitriol in dogs.

OBJECTIVE:To characterize oral bioavailability and pharmacokinetic disposition of etoposide when the IV formulation was administered orally to dogs.ANIMALS:8 tumor-bearing dogs.PROCEDURES:An open-label, single-dose, 2-way crossover study was conducted. Dogs were randomly assigned to initially receive a single dose of etoposide (50 mg/m2) IV or PO. A second dose was administered via the alternate route 3 to 7 days later. Medications were administered before IV administration of etoposide to prevent hypersensitivity reactions. Oral administration of etoposide was prepared by reconstituting the parenteral formulation with 0.9% NaCl solution and further diluting the reconstituted mixture 1:1 with a sweetening agent. Plasma samples were obtained after both treatments. Etoposide concentrations were measured with a high-performance liquid chromatography assay, and plasma etoposide concentration-time profiles were analyzed by use of noncompartmental methods.RESULTS:4 dogs had hypersensitivity reactions during IV administration of etoposide. No adverse effects were detected after oral administration. Plasma etoposide concentrations were undetectable in 2 dogs after oral administration. Oral administration of etoposide resulted in significantly lower values for the maximum plasma concentration and the area under the plasma etoposide concentration-versus-time curve, compared with results for IV administration. Oral bioavailability of etoposide was low (median, 13.4%) and highly variable among dogs (range, 5.7% to 57.3%). CONCLUSIONS AND CLINICAL RELEVANCE-Vehicle-related toxicosis can limit the IV administration of etoposide in dogs. The parenteral formulation of etoposide can be safely administered orally to dogs, but routine use was not supported because of low and variable oral bioavailability in this study.

BACKGROUND:The optimal treatment after inducing complete remission (CR) in dogs with lymphoma has not been established.HYPOTHESIS:After inducing CR with L-asparaginase, vincristine, cyclophosphamide, doxorubicin, prednisone (L-CHOP); consolidation with either half-body radiation therapy (HBRT); or lomustine (CCNU) and mechlorethamine, vincristine, procarbazine, prednisone (MOPP) would improve first remission duration compared with continuing a CHOP-based protocol for an additional 4 months.ANIMALS:Dogs with stage III-V lymphoma.METHODS:Prospective clinical trial in which dogs initially were treated with an 8-week induction protocol that consisted of L-CHOP. Dogs in CR after induction were then allocated to 1 of 2 consolidation arms. A chemotherapy consolidation arm consisted of 2 treatments with CCNU and 1 cycle of MOPP. A HBRT arm consisted of 2 sequential 8.0-Gy fractions to the cranial and caudal half-body separated by 30 days. Vincristine was given between fractions. Results of the consolidation arms also were compared with a historical group treated with the same 8-week induction protocol followed by CHOP therapy until week 24.RESULTS:Overall, 67% of the dogs were in CR after 8 weeks of induction chemotherapy and were compared. Fifty-two dogs were in the historical arm, 23 in the CCNU/MOPP arm, and 27 in the HBRT arm. No difference in first remission duration was found among groups. Median first remission duration for the historical, CCNU/MOPP, and HBRT arms were 307, 274, and 209 days, respectively (P = .28). Overall second CR rate was 82% and was not different among groups (all P > or = .58). Overall remission duration (P = .28) and survival time (P = .48) were not different among groups.CONCLUSIONS AND CLINICAL IMPORTANCE:Consolidation with either CCNU/MOPP or HBRT showed no advantage over a standard CHOP-based protocol.

OBJECTIVE:To determine the maximum tolerated dose and characterize the pharmacokinetic disposition of an orally administered combination of docetaxel and cyclosporin A (CSA) in dogs with tumors.ANIMALS:16 client-owned dogs with metastatic or advanced-stage refractory tumors.PROCEDURES:An open-label, dose-escalation, single-dose, phase I study of docetaxel administered in combination with a fixed dose of CSA was conducted. Docetaxel (at doses of 1.5, 1.625, or 1.75 mg/kg) and CSA (5 mg/kg) were administered concurrently via gavage twice during a 3-week period. Plasma docetaxel concentrations were quantified by use of high-performance liquid chromatography, and pharmacokinetic disposition was characterized by use of noncompartmental analysis. Dogs' clinical signs and results of hematologic and biochemical analyses were monitored for evidence of toxicosis.RESULTS:No acute hypersensitivity reactions were observed after oral administration of docetaxel. Disposition of docetaxel was dose independent over the range evaluated, and pharmacokinetic variables were similar to those reported in previous studies involving healthy dogs, with the exception that values for clearance were significantly higher in the dogs reported here. The maximum tolerated dose of docetaxel was 1.625 mg/kg. Gastrointestinal signs of toxicosis were dose limiting.CONCLUSIONS AND CLINICAL RELEVANCE:The absence of myelosuppression suggested that the docetaxel-CSA combination may be administered more frequently than the schedule used. Further studies are warranted to evaluate combination treatment administered on a biweekly schedule in dogs with epithelial tumors.