Immunohistochemical and transcriptomic characterization of T and myeloid cell infiltrates in canine malignant melanoma.

Immune checkpoint inhibitor therapy can provide significant clinical benefit in patients with certain cancer types including melanoma; however, objective responses are only observed for a subset of patients. Mucosal melanoma is a rare melanoma subtype associated with a poor prognosis and, compared with cutaneous melanoma, is significantly less responsive to immune checkpoint inhibitors. Spontaneous canine tumours have emerged as valuable models to inform human cancer studies. In contrast to human melanoma, most canine melanomas are mucosal-an incidence that may be leveraged to better understand the subtype in humans. However, a more comprehensive understanding of the immune landscape of the canine disease is required. Here, we quantify tumour infiltrative T and myeloid cells in canine mucosal (n = 13) and cutaneous (n = 5) melanomas using immunohistochemical analysis of CD3 and MAC387 expression, respectively. Gene expression analysis using the Canine IO NanoString panel was also performed to identify genes and pathways associated with immune cell infiltration. T and myeloid cell densities were variable with geometric means of 158.7 cells/mm2 and 166.7 cells/mm2, respectively. Elevated T cell infiltration was associated with increased expression of cytolytic genes as well as genes encoding the coinhibitory checkpoint molecules PD-1, CTLA-4, TIM-3 and TIGIT; whereas increased myeloid cell infiltration was associated with elevated expression of protumourigenic cytokines. These data provide a basic characterization of the tumour microenvironment of canine malignant melanoma and suggest that, like human melanoma, inherent variability in anti-tumour T cell responses exists and that a subset of canine melanomas may respond better to immunomodulation.

Incidence and risk factors associated with development of clinical cardiotoxicity in dogs receiving doxorubicin.

BACKGROUND: Doxorubicin (DOX) can cause cumulative cardiotoxicity in dogs, but the incidence of clinical cardiotoxicity in dogs receiving DOX has not been determined. HYPOTHESIS/OBJECTIVES: To determine if the duration of DOX infusion influences the incidence of cardiotoxicity, to characterize the incidence of clinical cardiotoxicity in dogs during or after DOX chemotherapy, and to identify any risk factors associated with cardiotoxicity. ANIMALS: Four-hundred ninety-four dogs that received at least 1 dose of DOX for the treatment of cancer. METHODS: Retrospective study of dogs that received DOX from 2006 to 2015. RESULTS: Of 494 dogs, 20 (4.0%) developed clinical cardiotoxicity. The duration of DOX infusion was not significantly associated with clinical cardiotoxicity, whereas a higher cumulative dose of DOX, higher body weight, decreases in fractional shortening after 5 doses of DOX, and development of ventricular premature contractions were significantly associated with clinical cardiotoxicity. High-risk breeds for developing dilated cardiomyopathy had an incidence of 15.4%, whereas low-risk breeds had an incidence of 3.0%. CONCLUSIONS AND CLINICAL IMPORTANCE: Although the duration of DOX infusion did not influence the incidence of cardiotoxicity, premature contractions and decreases in fractional shortening should raise concern for the development of clinical cardiotoxicity. Overall, the incidence of clinical DOX-induced cardiotoxicity is low, but Boxers and other breeds at high risk for dilated cardiomyopathy may be at an increased risk.

The role of cellular contact and TGF-beta signaling in the activation of the epithelial mesenchymal transition (EMT).

The epithelial mesenchymal transition (EMT) is one step in the process through which carcinoma cells metastasize by gaining the cellular mobility associated with mesenchymal cells. This work examines the dual influence of the TGF-ß pathway and intercellular contact on the activation of EMT in colon (SW480) and breast (MCF7) carcinoma cells. While the SW480 population revealed an intermediate state between the epithelial and mesenchymal states, the MC7 cells exhibited highly adhesive behavior. However, for both cell lines, an exogenous TGF-ß signal and a reduction in cellular confluence can push a subgroup of the population towards the mesenchymal phenotype. Together, these results highlight that, while EMT is induced by the synergy of multiple signals, this activation varies across cell types.

Serum 25-hydroxyvitamin D concentrations in dogs with osteosarcoma do not differ from those of age- and weight-matched control dogs.

Vitamin D concentrations show an inverse correlation with incidence of certain tumors in people and dogs. Additionally, human osteosarcoma has been associated with dysregulation of vitamin D-dependent pathways. The study objective was to compare serum 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 in 20 dogs with osteosarcoma to age- and weight-matched control dogs. We hypothesized that dogs with osteosarcoma would have lower serum 25-hydroxyvitamin D than control dogs. The mean 25-hydroxyvitamin D3 concentrations for dogs with osteosarcoma and matched-controls were 34.95 ng/mL and 33.85 ng/mL, respectively (P = 0.784). Based on these data, 25-hydroxyvitamin D insufficiency might not be important in the pathogenesis of canine osteosarcoma.

Heterotaxin: a TGF-ß signaling inhibitor identified in a multi-phenotype profiling screen in Xenopus embryos.

Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a pyridine analog, heterotaxin, which disrupts both cardiovascular and digestive organ laterality and inhibits TGF-ß-dependent left-right asymmetric gene expression. Heterotaxin analogs also perturb vascular development, melanogenesis, cell migration, and adhesion, and indirectly inhibit the phosphorylation of an intracellular mediator of TGF-ß signaling. This combined phenotypic profile identifies these compounds as a class of TGF-ß signaling inhibitors. Notably, heterotaxin analogs also possess highly desirable antitumor properties, inhibiting epithelial-mesenchymal transition, angiogenesis, and tumor cell proliferation in mammalian systems. Our results suggest that assessing multiple organ, tissue, cellular, and molecular parameters in a whole organism context is a valuable strategy for identifying the mechanism of action of bioactive compounds.

Feasibility of using gene expression analysis to study canine soft tissue sarcomas.

The prognosis given for canine soft tissue sarcomas (STSs) is based primarily on histopathologic grade. The decision to administer adjuvant chemotherapy is difficult since less than half of patients with high-grade STSs develop metastatic disease. We hypothesize that there is a gene signature that will improve our ability to predict development of metastatic disease in STS patients. The objective of this study was to determine the feasibility of using cDNA microarray and quantitative real-time PCR (qRT-PCR) analysis to determine gene expression patterns in metastatic versus nonmetastatic canine STSs, given the inherent heterogeneity of this group of tumors. Five STSs from dogs with metastatic disease were evaluated in comparison to eight STSs from dogs without metastasis. Tumor RNA was extracted, processed, and labeled for application to the Affymetrix Canine Genechip 2.0 Array. Array fluorescence was normalized using D-Chip software and data analysis was performed with JMP/Genomics. Differential gene expression was validated using qRT-PCR. Over 200 genes were differentially expressed at a false discovery rate of 5%. Differential gene expression was validated for five genes upregulated in metastatic tumors. Quantitative RT-PCR confirmed increased relative expression of all five genes of interest in the metastatic STSs. Our results demonstrate that microarray and qRT-PCR are feasible methods for comparing gene signatures in canine STSs. Further evaluation of the differences between gene expression in metastatic STSs and in nonmetastatic STSs is likely to identify genes that are important in the development of metastatic disease and improve our ability to prognosticate for individual patients.

A phase I trial of hyperthermia-induced interleukin-12 gene therapy in spontaneously arising feline soft tissue sarcomas.

Interleukin-12 (IL-12), a proinflammatory cytokine, shows anticancer properties. Systemically administered IL-12 causes dose-dependent toxicity. To achieve localized intratumoral gene expression, an adenoviral gene therapy vector with IL-12 controlled by a heat-inducible promoter (heat shock promoter 70B) was developed and tested in a phase I clinical trial in cats with spontaneously arising soft tissue sarcoma. A feasibility study was done in 16 cats with soft tissue sarcoma using murine IL-12 and/or enhanced green fluorescent protein adenoviral vectors under cytomegalovirus or heat shock promoter 70 control. Subsequently, we conducted a phase I clinical trial using an adenoviral feline IL-12 construct in 13 cats with soft tissue sarcoma. The soft tissue sarcomas were irradiated (48 Gy/16 fractions) followed by intratumoral injection of adenovirus. Twenty-four hours postinjection, tumors were heated (41 degrees C, 60 min). Tumor expression of feline IL-12 and IFN-gamma was determined. Cats were monitored for systemic toxicity. For the murine IL-12 construct, an association was noted between viral dose and murine IL-12 levels within tumor, whereas serum levels were minimal. Mild toxicity was noted at 10(11) plaque-forming units (pfu). With the feline IL-12 construct, high levels of feline IL-12 mRNA were detected in tumor biopsies with low or absent IFN-gamma mRNA following gene therapy. Hematologic and hepatic toxicities were noted at the highest viral doses and were associated with detection of IFN-gamma mRNA in tumor. It is possible to localize gene expression and limit systemic toxicity of IL-12 using the hyperthermia-induced gene therapy approach. The maximum tolerated dose of the feline IL-12 adenoviral vector was 10(10) pfu/tumor as dose-limiting toxicities were noted at the 4 x 10(10) pfu dose.

Phase I trial of doxorubicin-containing low temperature sensitive liposomes in spontaneous canine tumors.

PURPOSE: To determine the maximum tolerated dose, dose-limiting toxicities, and pharmacokinetic characteristics of doxorubicin encapsulated in a low temperature sensitive liposome (LTSL) when given concurrently with local hyperthermia to canine solid tumors. EXPERIMENTAL DESIGN: Privately owned dogs with solid tumors (carcinomas or sarcomas) were treated. The tumors did not involve bone and were located at sites amenable to local hyperthermia. LTSL-doxorubicin was given (0.7-1.0 mg/kg i.v.) over 30 minutes during local tumor hyperthermia in a standard phase I dose escalation study. Three treatments, given 3 weeks apart, were scheduled. Toxicity was monitored for an additional month. Pharmacokinetics were evaluated during the first treatment cycle. RESULTS: Twenty-one patients were enrolled: 18 with sarcomas and 3 with carcinomas. Grade 4 neutropenia and acute death secondary to liver failure, possibly drug related, were the dose-limiting toxicities. The maximum tolerated dose was 0.93 mg/kg. Other toxicities, with the possible exception of renal damage, were consistent with those observed following free doxorubicin administration. Of the 20 dogs that received > or = 2 doses of LTSL-doxorubicin, 12 had stable disease, and 6 had a partial response to treatment. Pharmacokinetic variables were more similar to those of free doxorubicin than the marketed liposomal product. Tumor drug concentrations at a dose of 1.0 mg/kg averaged 9.12 +/- 6.17 ng/mg tissue. CONCLUSION: LTSL-doxorubicin offers a novel approach to improving drug delivery to solid tumors. It was well tolerated and resulted in favorable response profiles in these patients. Additional evaluation in human patients is warranted.

Thermal dose is related to duration of local control in canine sarcomas treated with thermoradiotherapy.

PURPOSE: To test that prospective delivery of higher thermal dose is associated with longer tumor control duration. EXPERIMENTAL DESIGN: 122 dogs with a heatable soft tissue sarcoma were randomized to receive a low (2-5 CEM43 degrees CT90) or high (20-50 CEM43 degrees CT90) thermal dose in combination with radiotherapy. Most dogs (90%) received four to six hyperthermia treatments over 5 weeks. RESULTS: In the primary analysis, median (95% confidence interval) duration of local control in the low-dose group was 1.2 (0.7-2.1) years versus 1.9 (1.4-3.2) years in the high-dose group (log-rank P = 0.28). The probability (95% confidence interval) of tumor control at 1 year in the low-dose versus high-dose groups was 0.57 (0.43-0.70) versus 0.74 (0.62-0.86), respectively. Using multivariable procedure, thermal dose group (P = 0.023), total duration of heating (P = 0.008), tumor volume (P = 0.041), and tumor grade (P = 0.027) were significantly related to duration of local tumor control. When correcting for volume, grade, and duration of heating, dogs in the low-dose group were 2.3 times as likely to experience local failure. CONCLUSIONS: Thermal dose is directly related to local control duration in irradiated canine sarcomas. Longer heating being associated with shorter local tumor control was unexpected. However, the effect of thermal dose on tumor control was stronger than for heating duration. The heating duration effect is possibly mediated through deleterious effects on tumor oxygenation. These results are the first to show the value of prospectively controlled thermal dose in achieving local tumor control with thermoradiotherapy, and they establish a paradigm for prescribing thermoradiotherapy and writing a thermal prescription.

Chemotherapy followed by half-body radiation therapy for canine lymphoma.

A protocol of induction chemotherapy followed by half-body radiation therapy for treatment of lymphoma was used in 94 dogs. Seventy-three (78%) dogs achieved complete remission. Substage (P = .011) and phenotype (P = .015) were identified as predictors of complete remission rate. Of these, 52 dogs received half-body irradiation. Cranial and caudal halves received a total dose of 8.0 Gy, given in 2 fractions of 4.0 Gy on consecutive days with cobalt-60 photons and a 3-week interval between halves. Median 1st remission for these dogs was 311 days. Anemia was identified as the only predictor for length of 1st remission (P = .024). Toxicoses after half-body irradiation generally were mild and infrequent and included myelosuppression and gastrointestinal signs. Thirty-one dogs relapsed and 20 resumed treatment with induction followed by maintenance chemotherapy. Seventeen (85%) dogs achieved a 2nd complete remission. Median overall remission for all 52 dogs was 486 days. Results of this study suggest that half-body radiation therapy after induction chemotherapy is well tolerated and might increase remission duration compared with conventional protocols that use chemotherapy alone, but this increase might not be long enough to be clinically relevant or to justify application of the method described herein.

A retrospective analysis of 140 dogs with oral melanoma treated with external beam radiation.

Despite the early notion that canine oral malignant melanoma is radioresistant, recent data suggest that external beam radiotherapy is effective in local tumor control. However, optimal fractionation schedules have not been established. The high rate of regional and distant metastasis is another problem that has hindered long-term control. The role of chemotherapy in the management of canine oral melanoma has also not been determined. In this study, data from 140 dogs irradiated at North Carolina State University were evaluated with the following objectives: (1) to compare the efficacy of three radiation therapy protocols (36 Gy, 9 Gy x 4 fractions; 30 Gy, 10 Gy x 3 fractions; or >45 Gy, 2-4 Gy x 12-19 fractions) for the treatment of dogs with oral malignant melanoma, (2) to identify any host or tumor factors influencing prognosis, and (3) to determine the impact of systemic chemotherapy on treatment outcome. Information regarding response to therapy, disease progression, and survival were determined from the medical records or from information obtained by telephone or mail survey. Relationships between host, tumor, and treatment variables and outcome measures (response, time to first event, and survival) were evaluated using Fisher's exact test (response) and the Cox regression model (time to first event and survival). The median time to first event for the 140 dogs was 5.0 months (95% C.I., 4-6 months) and the median survival was 7.0 months (95% C.I., 6-9 months). In the univariate analysis, the following variables were associated with increased time to first event and survival: (1) rostral tumor sublocation; (2) lack of bone lysis observed on skull imaging, and (3) microscopic tumor burden. In a multivariate analysis of 111 dogs with complete data for these variables, tumor sublocation, bone lysis, and tumor volume were identified as joint predictors of time to first event (p < .001, p < .001, and p = .04, respectively) and survival (p < .001, p < .001, and p = .05, respectively). There were no differences in response, time to first event and survival between the three radiation therapy protocols used. Systemic chemotherapy had no impact on the development of metastatic disease, time to first event, or survival, although the dosages used in this study were suboptimal. External beam radiation therapy is effective in local disease control of canine oral malignant melanoma; however, the optimal fractionation scheme has yet to be determined. The high metastatic rate observed with this disease and the inefficacy of systemic chemotherapy indicate that further investigation into novel therapies is warranted.

Preoperative radiotherapy for vaccine associated sarcoma in 92 cats.

Medical records for 92 cats with a vaccine associated sarcoma receiving preoperative irradiation, with or without chemotherapy, between December 1985 and September 1998 were reviewed. The purposes were to quantify response to treatment and to attempt identification of factors associated with favorable response. Variables evaluated for a relationship to outcome included signalment, tumor location, presence of gross vs. microscopic tumor, radiation field size, irradiation technique, type of surgical procedure, completeness of excision, and chemotherapy (none, carboplatin alone, and others). Time to first event was calculated for the first day of treatment until local tumor recurrence or metastasis, or the date of euthanasia or death. Median time to first event for all 92 cats was 584 days. Only completeness of surgical excision was related to the time to first event. Median time to first event in cats having complete surgical excision was 986 days compared to 292 days for cats with incomplete excision (P = 0.004). Cats requiring bone removal to effect tumor removal had earlier failure than cats having other types of surgery. There was not a significant relationship between administration of chemotherapy or chemotherapy type and time to first event although outcome in cats receiving carboplatin was better than all other treatment groups. Carboplatin addition to preoperative irradiation appears worthy of further study. Preoperative irradiation is an effective treatment for cats with vaccine associated sarcoma, especially if complete excision can be accomplished following irradiation.

Prospective clinical evaluation of serum cardiac troponin T in dogs admitted to a veterinary teaching hospital.

The purpose of this study was to measure serum cardiac troponin T (cTnT) with a commercially available human enzyme-linked immunoassay (ELISA) test in various groups of dogs, including those undergoing doxorubicin chemotherapy. Serum samples were obtained from 6 groups of dogs: (1) normal adult dogs (n = 15); (2) dogs with asymptomatic dilated cardiomyopathy (n = 5); (3) dogs with congestive heart failure (n = 10); (4) dogs with untreated neoplasia (n = 20); (5) dogs with skeletal muscle trauma (n = 10); and (6) dogs with neoplasia receiving doxorubicin chemotherapy (n = 4). One serum sample was obtained from each of the normal dogs, those with asymptomatic cardiomyopathy, those with congestive heart failure, and those with untreated neoplasia. Serum samples were obtained serially from the dogs that were undergoing doxorubicin chemotherapy; samples were collected before doxorubicin (30 mg/m2) administration and then 1, 5, 7, and 14 days after administration throughout 6 cycles for a cumulative total dose of 180 mg/m2. All normal dogs, dogs with untreated neoplasia, and dogs with asymptomatic dilated cardiomyopathy had cTnT concentrations below the lower limits of detection for the assay used (<0.05 ng/mL). Detectable concentrations of cTnT were found in 3 dogs with congestive heart failure and in 2 dogs with skeletal muscle trauma. Detectable concentrations also were found in both dogs that had received 180 mg/m2 of doxorubicin. We conclude that dogs with congestive heart failure and those with skeletal muscle trauma and dogs with neoplasia receiving high-dose doxorubicin chemotherapy may have increased serum cTnT concentration, which may be suggestive of myocardial damage.